Lab Notebook

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  [[May|May]][[June|June]]
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  [[May|May]] [[June|June]] [[July|July]] [[Team 1|Team 1]] [[May 2007]]
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'''June 1''':
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*Team 3: Today we found an article on how osmotic shock affected one particular bacteria. If we are still continuing with the idea of osmotic shock, I think it would be a good reference article. Here's the link:  http://www.blackwell-synergy.com/links/doi/10.1111/j.1432-1033.1997.00572.x/pdf <br>Some interesting websites to revisit http://www.jbc.org/cgi/content/abstract/275/9/6047 <br>We were thinking of expressing luciferase and MscL in a Stable 3 cell, and then during the osmotic shock, as the water flows into the cell, there is a chance that luciferin will be able to enter the cell according to Elvis. So hopefully, the luciferin will then react with luciferase reslting in the emission of light. We think that this idea is worth a try. We found an article pertaining to the influx of solutes "Release of Thioredoxin via the Mechanosensitive Channel MscL during Osmotic Downshock of Escherichia coli Cells" by Bassam Ajouz, Catherine Berrier, Alexia Garrigues, Madeleine BesnardDagger , and Alexandre Ghazi§
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'''July 1''' Saturday
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*Hmmm, I hope people haven't stopped working just because I'm at the north pole... here at the north pole, we have stopped working for a bit, due to bad weather, and are sitting around eating, getting fat, and sending email. Quite the life. There's snow. Hope the ligations are working.
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*Team 3 (Cont'd)- We digested the pGFP vector with EcoR1 and Hind 111. The digestion was then run on a gel, the proper bands were obtained, and the vector was physically extracted.  
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'''July 3''' Monday
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*Team 1: Decided to cut what was isolated from midi-preps w/ BamH1. Going to run digests on a gel to see if we get characteristic bands. Primers should arrive tomorrow for YFP PCR. Hope you're having fun Jay!
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*Elvis-Horia: Chemically Competent MJF367 & MJF465 with pUC19 cell plates were very good (bacteria lawn); seeded each strain. Transformed MJF367-pGFP & MJF465-pGFP and plated.
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*Team 2: Set up screening digestion of E0422/C0012 ligation made master mix with the following quantities: 3.75 uL enzyme BsrbI, 7.5 uL Buffer2, 48.75 uL dH2O.  This was divded into 5 tubes and 3 uL of miniprep DNA was added to each. Ran a gel of the digested DNA and un-digested miniprep DNA and found that there was no DNA.  Did DNA precipitation following following protocol on 6/22/06.  Prepared screening digestion of the precipitated DNA.  Digested overnight.
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*Team 1: Today both the person responsible for half GFP's and the person responsible for sticky cells were e-mailed. No response fro Sticky cells and the GFP person was looking but did not find the plasmids. We were mostly researching today regarding a new Idea bout making a flagella fusion protein so it can attach to the membrane of another cell and forma a chain. We think that the only protein we can make a fusion of is the FliD. We were not successfull in asking the Microbiology departemtn about those fusion proteins, nor were we able to find this on the Internet as of current Date
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*Team 3: Ashwin and Jieun: New plating of the transformed Top 10F containing the ligation product was performed on two amp plates. For the future use, another ligation was performed using the same DNAs (Luc from June 21st, which had very strong bands on the gel analysis and GFP from Elvis) with the same ratio from June 21st.
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*Team 2: Dissolved the BBa_I5610 in 10 u'''L''' of dH2O, and then transformed dh5α with the plasmid. Jamie, Adam, and Aaron prepared more LB, and Aaron plated the the dh5α onto a plate using 2 u'''L''' on one half and 20 u'''L''' on the other half.
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'''July 4''' Tuesday
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*Team 3: Jieun: Transformation of ligation product was performed (one vial of Top10F with 10uL of ligation product). The other 20uL of ligation product is stored in 4C fridge for overnight, and the plates containing trasformed cells are kept in 37C incubator room (starting from 5:30pm EST).
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'''June 2''':
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*Seeded 2 colonies from pB10+Eco-Mscl in DH5a overnight.
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*1. Elvis has done the seeding of the GFP bacteria from the plates with MJF367-pGFP & MJF465-pGFP and placed it in the incubator. After ~6h30 incubating, Horia added 1uL IPTG and placed in incubator for another hour. <i>Update:</i> The cells showed minimal Green Fluorescence under the UV wand. 2.The control of the MJF-pUC19 seeding was cloudy; the seeding were thrown away.  
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*Team 1: Because the person with the half GFP coluld not find the plasmids. We needed to get the mammalian plasmids from upstais to make a recombination to bacterial expression plasmids.
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*Team 2: Ran a gel of our precipitated DNA and digested DNA. Found there was no DNA.  Seeded more colonies of the E0422/C0012 ligation.
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*Team 2: There were three colonies on the plate of Top10 transformed with I5610. All three colonies were seeded. Dissolved I15004 and R0062 and transformed Top10 with them.  Both I15004 and R0062 were plated.
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*Team 1: Did three seedings with 2 controls in 2 mL. Digested Fos-Beta a second time. Ran Fos-Beta on a gel but had no DNA in our lane. At 5 PM, added more LB to our seedings and left them overnight in incubator.  
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*Team 3: We extracted the GFP vector from the gel in the tube labeled "GFP by HIND 111 EcoR1 June 1,2006". The tube containing the purified DNA GFP vector from the gel is labeled "Gel extraction of GFP vector 6/2" A gel was then run to screen the GFP vector as well as the lucferase genes. Well #1 had the DNA ladder, Well #3 is the DNA from the "Gel Extraction of GFP vector 6/2", Well #4 has the "luciferase gene eluate May 10, 2006", Well #5 contains DNA from "lucif gene 18/06".
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'''July 5'''
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*Team 2: Miniprepped seedings from yesterday, followed protocol on May 9, 2006. Set up screening digestion of miniprepped DNA.  Used the same quantities as on July 3, 2006.  Ran a gel of this digested screening and undigested miniprep. All 5 samples had DNA in them.  Unable to tell if the DNA was cut properly because the lanes were smeared significanly.  Made new TAE IX Buffer in an attempt to fix the constant smearing.
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*Team 1: Out of 3 seedings, only one grew. Did 3 mini-preps out of that one seeding. Ran 10 microliters of one miniprep in a gel to test if we got DNA. Nothing showed up. Decided to go back to culture plates. Transferred bacteria from one plate onto another amp plate to test if they are truly amp resistant. Put in 37 degee incubator overnight. Received primers for PCR! Will check plate tomorrow. If colonies grow, we're good. If not, need to consider redoing transformation.
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*The gel was analyzed and the GFP vector did not show up on the gel. However, both luciferase bands appeared at the appropriate site. The file was saved as "jun 2- GFP,luc gene" in the iGEM folder.
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'''July 6'''
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*Team 1: We are good, we got colonies! Yay! Picked 10 off plate and did 10 seedings, each in 2 mL. Also did two digests, one of Fos-Beta DNA and one of miniprep from colonies off original pDsBiFC plate. Also just ran non-digested mini-prep DNA to test hypothesis if we are losing DNA with restriction digests. Ran a gel. The Fos-Beta showed up beautifully with the right size bands so now we know we have isolated both the Fos-Beta and Jun-Beta plasmids! However, no DNA showed up in the lanes for the miniprep. That's ok though because we have 10 new seedings. Came back in the evening to find that all 10 seedings grew and the controls were clear. Spun 10 tubes down and resuspended with P1. In the end, had two tubes of resuspended cells. Saved cells and culture tubes in fridge.
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*Team 2: Did large scale digest for each of the 5 miniprep samples from yesterday.  Expected bands at 1800 bp and 2030 bp.  Ran gel of digests and had a band at 1800 and 500 bp.  Came to the conclusion that the ligation product did not work properly.  Screening for E0422 original plasmid minipreps: Cut with BsrBI.
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NB: Two dH2O bottles smelled like Romanian toilets, so I threw them out and placed the remaining bottles under the pcr enclosure.
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'''July 7'''
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*Team 2: Ran gel of E0422 screening digests.  Expected bands at 1800 and 1200 bp, well 1 had band at 1800, well 2 had band at 2000, and well 3 had band at 1800, 1600, and 1200 bp.  Transformed 2 uL of E0422 brick supplied in homemade Top10 cells.
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*Team 1: Completed 2 minipreps from the cultures that grew last night. Then did 2 digests of miniprep DNA w/ EcoRI and BamHI and ran a gel of those digests in the afternoon. Purpose was to screen PLasmid DNA to make sure it is the pDsBiFC plasmid that we want.
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'''June 4 evening:  
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'''July 9'''
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*Team 2: Seeded 4 colonies from transformation plate into LB + Amp for miniprep.
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*Elvis (the king) seeded MJF465-pGFP cells in 1mL LB with AMP
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'''July 10 Monday'''
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*Team 1: Results from gel that was run on the 7th were not good because it seems the enzymes did not cut. Decided to run three more digests, 2 single ( 1 EcoRI and 1 BamHI, and 1 double w/ both of them) We then ran all digests on the same gel and we got our expected results! Yes! The only problem is the bands were so faint that you could barely see them.
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*Team 2: Miniprepped 5 mL of broth culture for each column.  Prepared screening digests with BsrBI according to proportions listed on July 6/06.  After running on gel, found only sample 4 showed a band, and it was at about 2000 bp.  Annette recommended screening with a different enzyme (NotI).  Set up screening digest with NotI.  Run gel of digests with samples of today's miniprep to see if any DNA present in these minipreps.  Well 4 was the only well to contain the correct DNA (band at 3000, 2000, and 1000 bp).  Well 6 and 7 contained DNA but the identity could not be confirmed.  Well 5 did not contain DNA, and wells 1, 2, and 3, did not contain the correct plasmid.
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'''June 5''':
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'''July 11'''
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*Team 2: Complete a large scale digest of the sample run in well 4.  Cut using EcoRI, XbaI, and ScaI.  Realized that this was very bad, because it destroyed our plasmid.  Oops.  Re-did the large scale digestion using EcoRI and XbaI, digested for 4 hours.  Added 3 uL of CIP, incubated at 37C overnight.
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*Jieun diluted the MJF465-pGFP seeding into 5mL LB. After 4 hours, Horia verified O.D. with 1mL of culture to obtain a value of 0.25 and replaced them in the shaker-incubator. One hour later, 4uL of IPTG was added and left in S-I for Jamie to pick up later (~7:30pm).
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'''July 12'''
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*Team 2: Performed DNA desalting to remove enzymes and small DNA fragment removed between EcoRI and XbaI sites. 53 uL sample volume -> 159 uL QX1, 3.8 ug DNA -> 10 uL QiaexII. Need to ligate C0012 brick (cut at EcoRI and SpeI) into E0422 with plasmid (cut at EcoRI and XbaI). Did calculations for ligation, set up ligation of E0422 and C0012, transformed into commercial Top10, and plated.
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*Team 1: We prepared competent cells Top 10F'. We have also ordered primers for the Jun-YFP and Fos-YFP. The following our our primers.
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'''July 13'''
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*'''bJunYN155'''
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*Team 2: Seeded 8 colonies from ligation plate for miniprep next day.
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*Forward: 5'-CACCGTGTACGGTGGGAGGTA-3'
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*Backward: 5'-ACTGGGGAGGGTCACAG-3'
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*'''bFosYC155'''
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'''July 14'''
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*Forward: 5'-CACCTTCTAGGCCTGTACGGAAGTG-3'
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*Team 2: Miniprepped 8 seedings, using 5 mL of broth culture for each miniprep. Screened the minipreps using Not I and Sca I, expecting bands at 2kb, 1500 bp, and 500 bp. Incubated for a couple of hours. When gel ran, observed bands at 4 kb, and a wide band at 3500 kb - perhaps the plasmid was only cut once, and the remaining uncut plasmid caused the wide band.
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*Backward: 5'-GACCATGATTACGCCAAGCTA-3'
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*Team 2: The plates of I15004 and R0062 turned out well.  The controls of the seeding done on June 2, 2006 did not turn out well.  Seeded two colonies of I15004 and R0062 and replated the refridgerated DH5α that was transformed with I5610. Dissolved E0422, B0034, and C0012.  E0422 was placed in DH5α, and B0034 and C0012 were placed in Top10.
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'''July 16'''
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*Team 2: Set up screening digests for 4 of July 14 minipreps, again using Not I and Sca I in case the problem was due to lack of incubation time. Incubated overnight.
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*Team 3: We re-digested pGFP (from May 29th's midiprep) with EcoRI and HindIII. The gel was slightly smeared and the band for the GFP insert was very dim. But there was a visible band of pGFP vector (with no insert), which was cut out. The Gel Extraction procedure was then carried out, and the tube was labeled "Purified pGFP 06/05", and stored in the freezer in spot 1A of Cloning Fragments Box #1.
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'''July 17'''
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*Team 2: Gel ran of last night's digests showed no DNA, and smeary ladder. Jay suggested our minipreps may not be clean enough, so we washed them with Buffer PB. New screening digests with same enzymes, ran gel and found that there was no ladder shown! Replaced TAE buffer - maybe it's eating our DNA. New screening digest set up for overnight incubation with Ase I.
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'''June 6'''
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'''July 18'''
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*Team 1: Absolutely nothing was done today as our primers did not arrive yet.
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*Team 2: Ran gel of last night's digests, expected bands at 1920 bp and 2200 bp. Observed band at 3000 bp. Set up another screening digest of all 8 minipreps from July 14, using Not I and Eco RV.  
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*Team 2: The replating of the refidgerated DH5α transformed with I5610 did not work. The plates with E0422, B0034, and C0012 worked well. The seeding of I15004 and R0062 went well. E0422, B0034, and C0012 were seeded. Top10 was transformed with I5610. I15004 and R0062 were miniprepped. Dissolved I15016 and Q04121 and then transformed Top10 with them.
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*Team 3: Team 3 is making its triumphant return after a two week hiatus from the lab notebook!!! We first prepared a concentrated miniprep of the psp-luc+ by using 250uL of Buffer P1 to    resuspend all 5 cell pellets. This miniprep was then digested with EcoRi and Hind 3 (digest labeled "psp-luc+mini prep conc 7/18"). The digested miniprep is placed in Cloning Fragments 6D and 6E. The "conc mini psp-luc+ 7/14" was also digested. The psp-luc+ and pGFP which had been digested yesterday was run on a gel. The gel was successful and the GFP vector band was seen at 3300bp while the luciferase gene band was seen at 1700 bp. The vector and the gene DNA was then extracted from the gel. The extracted DNA, the GFP vector "GFP 7/18" was placed in 6G and the luc gene "luc gene 7/18 was placed in 6F  :D
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*Team 3: A screening of the purified pGFP vector was done to reveal that no DNA was present. We did another digest of the whole pGFP plasmid with EcoRI & HindIII, ran it on a gel and cut out the fragments corresponding to the cut pGFP, which were placed in the freezer (in 4 microcentrifuge tubes).  
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'''July 19'''
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*Team 3: The "luc mini 7/17" and "luc mini conc 7/14" digests were screened on gel, both yielding faint luc gene bands (1.7kb). The ligation of "luc gene 7/18" and "GFP (vector) 7/18" was performed: 15uL insert, 2uL vector, 2uL lig. buffer, 1 uL ligase. Incubated for 2 hrs at rm temp. 2 vials of Top10F' (1 commercial and 1 homemade) were transformed with the ligation, and plated.
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* The induced MJF465-pGFP showed happy bacteria swimming but absolutely no expression of GFP under the uber microscope in Jay (z)'s lab.
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'''July 20'''
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*Team 3: Yesterday's ligation plates were removed from 37C and placed at 4C (at 11:00). The plate with homemade transf. Top10F' had no colonies, but the 2 plates with commercial transf. Top10F' had medium-sized colonies. To be seeded at 16:00.  
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*Elvis (the king) plated 40uL of the culture on chloroamphenicol plates to verify its identity.
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'''July 21'''
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*Team 3: A miniprep was prepared of the succesful seedings of the ligated GFP vector and the luciferase insert. The miniprep has now been digested to make sure that the DNA is in fact the ligated GFP and luciferase insert
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'''June 7'''
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'''July 25'''
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*Team 1: Finally our PCR primers have arrived, therefore we went upstais and prepared a PCR with the following procedure (important for future PCR reference for any team willing to do succesfull PCR.
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*Team 3: The digestion result revealed a few very important things. 2 minipreps out of 4 having been digested showed bands at 10kb and 8kb, and the other two at 4kb. The latter ones might contain the desired ligation product with the possibility of Sca1 inefficiency. The expected result from the digetion was 2300kb and 1900kb bands (adding up to approx 4kb, shockingly similar to the single bands that appeared on the gel). The minipreps of those latter ones are stored in the cloning fragment box (approx 30-50uL in each tube). A link to the paper about mechanotransduction activity in living cells has been posted under Journal Club Paper. Further analysis will be available after the group disccusion once Jieun snaps out of whatever she's going through.
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'''PCR ''' (by Adam):
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'''July 25'''
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*Team 3: The 6 ligation minipreps (prepared yesterday) were digested with ScaI, and screened on gel. Again strange (very faint) bands appeared, this time above the DNA ladder. Possibilities are that these bands come from garbage DNA (contamination or chromosomal DNA), or ScaI isn't slicing properly! We should screen the minipreps to see if they actually any useful DNA, and try digesting with another enzyme.
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Step 1: Take the DNA that is 1.28 ug/uL and make a 1000 fold dilution by taking another tube, adding 1000 uL PCR water and adding 1 uL of DNA. Mix Gently by inverting.
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'''July 27'''
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*Team 3: '''News To Horia:''' The Day Of Truth Has Arrived. According to Jieun "the truth is out there in the biohazard box." (long story- Jieun..was being herself = moody and Ashwini was being calm and rational) This time we digested with our best-buddies - EcoR1 and Hind III- and they did not dissappoint. The time had come, and the gel was placed on UV machine with Elvis' shaking hands. Jieun pressed the "Acquired" button, and voila. Did I say Voila? VOILA. BEAUTIFUL TWO SEPARATE BANDS PLACED WHERE THEY WERE SUPPOSED TO BE. We embraced this joyful moment by excessive hugging time and Jieun, despite her cranky and emotionally detached self, gave a big hug to her teammate,Ashwini. So, long story short. WE SUCCEEDED. BOOOOYAH. OHHHHH SNAPPPP!!!!
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Step 2: The primers arrive in a form that is '''DRY'''. Read on the label how many nanograms of DNA there are and add 10X as many uL of '''EB solution''' and making a 0.1 ng/uL solution. For example if 26.1 ng DNA, we add 261 uL water. After water addition the extra primers can be stored in the Freezer just like all other DNA.
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'''July 28'''
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*Team 2:  WORKING LIGATION!!
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Spep 3: A PCR tube is taken and the following additions are performed exactly '''in the following order'''. There is a P2 pipette available upstais if one has to use it.
 
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*A: 80 uL '''PCR''' water
 
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*B: 10 uL PCR buffer
 
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*C: 4 uL dNTP's
 
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*D: 2 uL each Forward and back Primer
 
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*E: 1 uL BSA
 
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*F: 1 uL Taq polymerase (Important Note: Take it only when absolutely ready for it. It is tasken out of the fridge and into the portable freezerbox only when the things above have been added. After use it must immediately return to its place in the freezer.
 
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*G: 2 uL DNA, the Diluted solution
 
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Step 4: The PCR tube is placed at the centre of the rack in the PCR machine and program 84 is turned on. Under no circumstances can one attempt to reprogram the machine.
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'''July 29'''
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*Team 2: The I15016 plate had massive growth. It needs to be transformed and plated again.  C0013 and B0034 seeds did not show any growth.  E0422 was successfully seeded.  Aaron and Jamie made amp plates.  E0422 was miniprepped.
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Notes from Jay:
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*Team 1: Adam worked till late hours purifying the gel pieces, but didn't ligate because the ligase was locked upstairs and we're out.
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Will anyone from Team 1 be in this weekend? I can do it if you like? Please email me if you're Team 1 and want to do something this weekend, I will be downtown this afternoon.
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To everyone, notes about the new Gel Purifcation Kit: please use ONLY ONE column for each piece that you cut out, no matter how much it weighs (try to minimize excess agarose).  You can spin more than once.  We only get 50 columns in the kit so please don't waste!  Also, if you're purifying a piece with a molecular weight close to that of the loading dye (either the green or the purple), you will get some loading dye into your agarose and this will turn your melted piece greenish or purplish.  This is OK and doesn't mean that you need to add sodium acetate.  In fact, I have never once in many many many hundreds of these had to add sodium acetate.  Always do the isopropanol addition, and the optional wash isn't necessary.
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C'est tout, bonne fin de semaine!
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*Team 3: Gel extraction of 2 of the pGFP vector tubes was performed. We had realized the protocols were not followed properly and less DNA were put into the tubes during previous extractions. The screening was good (bands visible at ~2.8kb. The remaining tubes of the purified DNA contain the dye 6x.(31.7uL pGFP + 6.3uL dye remains in each). Non-purified gel pGFP still remains in another 2 tubes in the freezer.
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'''August 9'''
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*Team 3: To do the luciferase protein assay, we don't need to lyse the cells to react luciferase with luciferin. Jay found a procedure which can be found here: http://www.btci.org/k12/bft/bgt_background.html
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*Elvis (the king) and Horia seeded 10 tubes with colonies from the chloroamphenicol plates growing MJF465-pGFP? in 1mL LB + 1uL AMP to make sure they contain pGFP.
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Our present goal is to make LAR (luciferase assay reagent - 1 mM luciferin in 100 mM sodium citrate pH 5.5). We will make the luciferin and sodium citrate solutions separately. The overall plan is to prepare a seeding (in LB) of our ligation cells with O.D. of ~0.3, induce it with IPTG, centrifuge, dissolve the pellet (cells) in LAR, and thence observe the expected bioluminescent reaction.
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*Alex prepared 2 gels for our PCR products tomorrow.
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'''August 10'''
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*Team 3: 500 mL of 100 mM sodium citrate (pH 5.5) was made yesterday and store at 4C. Today, we diluted a seeding of pGFP-luc cells (made on Aug. 8 by Ashwin) 1:150 - 600 uL seeding in 30 mL fresh 1X LB with 30 uL. Placed in shaking incubator until O.D. measured to be 0.365. 30 uL IPTG added to cells (1 uL IPTG/1 mL total volume), and placed in shaker for 2 hours. Also found that luciferin can enter E. coli without need of MscL channel ...
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'''June 8''' Thursday
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'''August 23'''
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*Team 1: Both our PCR products from the 7th had strange clumps that we could not explain. We ran our PCR product from the 7th on a gel. It was found that there were 2 bands for the Jun, and 2 extremely faint bands for the Fos. We decided to do a Gel extraction on both the Jun and the Fos.
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*Team 3: The new plates with pGFP-Luc had somewhat less of a lawn than the previous ones. A miniprep screening digest was performed by Ashwin - with similar results as the previous screening done by Alida. It seems the bands of DNA move more slowly than the ladder, and the bands are thick and almost but not exactly in the right place. Please refer to the gel photo. New seedings have been done from the new plate.
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*Team 3: Unpurified pGFP gel was purified into "GFP vector #1 6/8) and purified pGFP with dye in it(yes, I know) was run on the gel and was purified into "GFP vector #2 6/8." Both tubes were put into the freezer for screening tomorrow.
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'''August 24'''
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*Team 3: Beetle Luciferin is scheduled to arrive tomorrow (Friday). A seeding of ligation cells - diluted in fresh LB at an O.D. of 0.3-0.4 and stimulated with IPTG - must be spun down. Discard the supernatant, and the pellet of cells must be resuspended in 3-5 mL of the prepared sodium citrate. Transfer 1-1.5 mL of this solution to a microcentrifuge tube, and add ~0.25 mg of luciferin. The bioluminescence must be viewed in a dark room. The quantities described above (determined by Elvis and Ashwin) are estimated and may not be totally accurate.
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*The seedings of MJF465-pGFP showed no cultures, so Elvis (the king) & Horia prepared double antibiotic plates with Amp+Cam (chloroamphenicol) to select specifically for the MJF465 strain containing pGFP (naturally Cam resistant MJF cells may not have replicated pGFP when growing on Cam since they do not need it to survive). MJF465-pGFP & MJF367-pGFP cultures were plated by volumes of 10uL and 30uL.
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'''June 9''' Friday
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*Team 1: The Gel extraction from the 8th was tested on a Gel and it was found that there was some product for Jun but none for Fos. Therefore Julia and Belinda cloned the Jun into a Topo vector and transformed the topo colonies. They plated 150 uL on one plate and 100 uL on other platew to be picked up on the Saterday. Meanwhile Adam and Alex prepared a second PCR upstairs of the FosYFP to be left overnight.
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*Team 2: Transformed Top 10 with B0034.  Seeded I15016 and C0012.
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*Team 3: Screening of "GFP vector #1" and "GFP vector #2" (from 6/8) was positive, though the bands were faint. The ligation of pGFP to luc+ was not carried out, since we were not sure of the concentration of our luc+ sample from 5/10. A screening of the luc+ sample and our purified pGFP vector side-by-side will allow us to estimate the ligation ratio required (to be done Monday).
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*The MJF plates had only 3 colonies in total, which we baptized "super insane colonies". These were seeded, altough we do not expect them to be any good. A new transformation of MJF cc cells with pGFP was done. This was plated mistakenly on Amp plates by Horia, instead of Cam+Amp plates.
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'''June 12''' Monday
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*Team 1: The colonied from the 9th were good, therefore the Topo cloning worked. Seeding of 2 colonies (one from each plate) were done. The FosYFP PCR product from Friday was taken and placed on a Gel with 3 wells. To the 100 uL total product, 20 uL loading dye was added and this was copmpletely spread into 3 wells. It was found that the PCR product had worked very well and the bands were bright. These 3 wells were extractected and at the end ran on aother Gel, lane 6 of a common gel. Colonies were picked up again at 8:00 and the first steps of a miniprep were done including the first spin and placing in P1 buffer.
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*Team 2: The plating of Top 10 with B0034 did not work, but the seeding of I15016 and C0012 did.  Miniprepped I15016 and C0012.  Transformed Top 10 with B0034.  Digested E0422 with EcoRI and XbaI.  Digested C0012 with EcoRI and SpeI.  Ran the digests on a gel, but the gel results were poor.  Seeded I15016 and C0012 again.
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*Team 3: We screened luciferase from May 18th in the morning to realize the DNA was too old, hence no band was shown. Later, the digestion of pspLuc May 18th was carried out and another screening was done. Yet again, there was no band. The most likely suspect to this must-end-mystery was the miniprep procedure/kit. Seeding of pspLuc from the plate was done (2 controls - one with amp and one without amp, 2 regular seedings). Whether miniprep kit has problems will be determined tomorrow morning, and until then our seeding tubes have been soundly being shaken in the 37C incubator for overnight.
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*The seeding of the "super insane colonies" from June 9 seemed good, and they were transferred into fresh LB and left overnight. The transformant MJF+pGFP cells were plated again on Cam+Amp cells (50uL) and left overnight.
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'''June 13''' Tuesday
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*Team 1: The results from yesterday's Gel revealed that the FosYFP PCR product gel extraction worked and we began cloning this into a Topo vector. After cloning we did a Transformation of the Top10 cells provided by Invitrogen in the Topo kit. We then plated them on 2 plates, one used 100 uL and the other 150 uL and left for incubation. THere was a miniprep that was done on the Jun as well. We also mapped the Jun plasmid and found that to confirm , one could digest with XbaI. A small scale digest was done using the following values: 7.5 uL dH2O, 1.5 uL buffer 2, 5 uL DNA and 1 uL XbaI.This was placed on a Gel that was common with other groups. Results were viewed the next day.
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*Team 2: Jay-Z performed a miniprep to make certain the miniprep kit worked.  It did.  The plating of B0034 worked this time.  Repeated the miniprep of I15016 and C0012 as well as the digest.  Some of the miniprep was run on the gel, and both showed up.  Ran C0012 on a gel.  Clear bands showed up.  Extracted C0012 from the gel.  Heated the E0422 to 65°C to deactivate the restriction enzymes.  Added CIP to the E0422 to dephosphorylate the fragment overnight.  Seeded B0034.
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*Team 3: The seeding of pspLuc was successful. We were informed that there was no problem with miniprep kit (based on the comparison result between the old miniprep kit and the new one), and miniprep was carried out. Digestion of pspLuc miniprep was done, and the digest was run on the gel later in the afternoon. Special thanks to team 2 members, we were told that there were two bands around 1200 and 2000 basepair regions, indicating the presence of luciferase gene (approx 1700bp). The gel fragments have not been cut out yet, and the gel is stored in the fridge/freezer for tomorrow's extraction. Woohoo!
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*The MJF double antibiotic plates were picked from and seeded: 5 tubes for MJF367 and 3 tubes for MJF465. The other cultures of "SIColonies" were tranferred to 6mL LB and grown to O.D. 0.671, then induced with 5, 10 and 15 uL of IPTG. A few hours later, lo and behold at their power, earthling, the colonies expressed a very sexy amount of GFP! But not as sexy as YOU.
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'''June 14''' Wednesday
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*Team 1: Colonies were found on both the Fos plates that contained 100 uL and 150 uL plated with corresponding rough amounts of colonies. The news from the Gel was such that there was at least onepiece at 6000 bP and one at 1500 bP. The one at 6000 was initially though wrong but the plasmid map was updated to include certain details and the impropved map showed the increased size of 6000 bP. The disappointment was in the 1500 fragment which was actually supposed to be at 586 bP. It was decided that we do a double digest with SacI and SmaI as that will cut out our insert more precisely. The following values were used: 6.5 uL dH2O, 5 uL DNA, 1.5 uL buffer 4, 1.5 uL SacI, and 1.5 uL SmaI. The gel was again disappointing in that it only showed one band. After extensive staining and destaining it was found that there was a very faint smaller piece but too faint to be confirmed. Seeding of 2 colonies (one from each plate) of the TopoFosYFP was done at the end of the day.
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*Team 2: Extracted the E0422 from the CIP solution.  Calculated the volumes of E0422 and C0012 needed for ligation.  Ligated E0422 and C0012 and prepared a control (E0422 + ligase).  Miniprepped the B0034.  Digested B0034 with EcoRI and XbaI.  Used REact 3 as a buffer since we were out of EcoRI buffer.  Digested R0062 with EcoRI and SpeI.  Heated B0034 to 65°C for about 10 minutes to deactivate the restriction enzymes.  Ran R0062 on a gel, but nothing turned up.  Digested R0062 again with more miniprep.  Transformed Top 10 with the ligated E0422 and C0012 and plated the bacteria.
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*The seedings from June 13 showed cultures in the three MJF465 tubes only. These were transfered and grown in 4mL LB. At an unknown stage of growth they were induced with 2uL/mL, 1uL/mL and 0.5uL/mL IPTG and incubated for 2 hours. The stage of growth is unknown because the O.D. reading was done at 470nm not 600nm. All three tubes showed GFP.
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*Elvis (the king) has left the building. Until Friday.
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'''June 15''' Thursday
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*Team 1: The Seedings for Topo-FosYFP from June 14 were good and the controls were clear. 1.5 ml from each culture was utilized to do 2 minipreps into 50 uL dH2O. Two digests (one of the Fos Miniprep and one of the Jun Miniprep)were done using XbaI with the following values: 7.5 uL dH2O, 5 uL DNA, 1.5 uL Buffer 2, 1 uL XbaI. The gel took all 18 uL into one well and the results only showed a band at 6000, no smaller band. More miniprep seedings were done 5 of each Jun and Fos.
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*Team 2: Transformation of E0422/C0012 ligation was successful.  Seeded 3 ccolonies from this plate.  Ran a gel of R0062 digest, and did not find anything.  Another cloning stragegy was developed where the B0034 plasmid would be cut at Spe and Pst and the ligated C0012/E0422 plasmid would be cut at Xba and Pst.  This 3 piece brick set would then be cut at Spe and Xba and the R0062 plasmid would be cut at Spe and Pst.
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'''June 16''' Friday
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*Team 1: Massive minipreps from the seedings were done 5 for Fos and 5 for Jun
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*Team 2: All seedings from yesterday grew.  Prepared mini-preps; 2 from 2 different colonies. Followed protocol from May 9, 2006.  Combined 50 uL from each colony into 2, 100 uL samples.  Performed restriction digest using the following quantities:
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E0422/C0012: 22 uL dH2O; 15 uL E0422/C0012 miniprep; 5 uL BSA; 1.5 uL SpeI; 1.5 uL PstI
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B0034:  22uL dH2O; 15 uL B0034 miniprep; 5 uL Buffer 2; 5 uL BSA; 1.5 uL SpeI; 1.5 uL PstI
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Ran get of the C0012/E0422 digest product.  No DNA appeared on the gel.  Performed minipreps for 3 more colonies from the C0012/E0422 seedings following protocol on May 9, 2006.
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'''June 19''' Monday
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*Team 1: A digestion was performed of the first miniprep of each (hence 2 digestions) using XbaI. The amounts were 12.5 uL DNA, 1.5 uL Buffer 2, and 1 uL XbaI. The digest was for 45 minutes. The results revealed that the Jun miniprep digest had only one piece that corresponded to 6000 base pairs which implied it was not digested. The Fos was strange, however. There was a piece at 3000 base pairs which did not correspond to the size we needed that would be 1500 base pairs and the rest of the plasmid at 6000 base pairs did not show up. Apparently according to the professor, this miniprep looked to be a crazy recombinant. Therefore what was done next is a buinch of digestions of different minipreps with various restriction enzymes that were mapped on Friday. So we did 4 digest. One of each Jun and Fos with SacI (buffer 1, yellow ribbon) and HindIII (buffer 2, blue ribbon). We then left these digests overnight. The Minipreps used for the digests were, the ones numbered 2 in both cases for the SacI and the ones numbered 3 for HindIII. The same amounts were used, 12.5 uL DNA, 1.5 uL buffe, and 1 uL enzyme.
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*MJF465+pGFP 1mL seeding were diluted to 10mL volume and incubated in SI. O.D. was taken periodically. They were placed in fridge at 6PM at O.D. ~ 0.1. MJF465cc & MJF367cc cells were transformed with both pGFP and Eco-MscL and plated on double antibiotic plates.
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*Team 2: Did a restriction digest of the E0422/C0012 miniprep following protocol on May 3, 2006, using the following quantities: 22 uL dH2O; 15 uL E0422/C0012 miniprep; 5 uL BSA; 1.5 uL SpeI; 1.5 uL PstI.
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Made 15 uL digests of E0422/C0012 miniprep to fun for screening:  5uL miniprep of DNA, 1 uL each of XbaI and PstI, 1.5 uL BSA, 1.5 uL Buffer3, 5 uL dH2O.  Incubated at 37 C overnight.
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'''June 20''' Tuesday
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*Team 1: The digests from yesterday were ran on a gel and it was found that very little DNA was present. None was found in either of the SacI digests and a very small bit was found for the HindIII digest. What was found appeared to be a 6000 base pair piece for the Jun and a smaller piece (unkown because the ladder in this gel was all smeared up) for the Fos, but still only one piece for each lane as if there was no digestion. Apparently, due to a small misunderstanding and miscomunication, thagt was not what we were supposed to do with the massive minipreps. Apparently they should have each been screaned with the same restriction enzyme to see which ones are the right plasmid and which ones are crazy recombinhants. So essentially we should have done a massive digest with XbaI for all the minipreps in parallel. Because we were questioning if we had any DNA in the minipreps at all we decided to do a screan of all of them without a restriction digest, just 5 uL DNA and 1 uL dye. So we ran a gel like that, all 10 minipreps and found that all had DNA and the firs four of the 5 Jun minipreps seamed good and only miniprep 2 and 5 was good, the rest were smaller or larger fragments and there were 2 pieces in each corresponding to the Nicked and Supercoiled, one significantly brighter than the other.
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*Team 2:  Ran gel of our 5 miniprep digests.  Nothing found on the gel.  Not sure of the problem.  Seeded 6 new colonies from the E0422/C0012 ligation.  Tomorrow Jay will perform the minipreps and run the gel.
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'''June 21''' Wednesday
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*Team 1: Massive digests with Xba1 were done of all minipreps. This was performed by the prof using 3 uL DNA, 1.5 uL buffer, 0.5 uL XbaI and 10 uL dH2O. It was found that 2 of the fos minipreps showed 2 bands and 1 jun miniprep. However the size of the smaller bans were doule what they were supposed to be, therefore we decided to do digests of those minipreps with various other enzymes. Message from Alex: The Digests prepared by Belinda and I are ready to be run in a gel tomorrow morning. They are in a rack in the freezer. Also: we received the spanish DNA in the form of bacteria in two small tubes (and a third random one). The bacteria are in agar and I plated them on CAM plates freshly made by moi. The tubes are in the fridge and the plates are in the 37c room. I will be there tomorrow at 10 (with Adam I believe) to run the gel and look at our plates: Adam: if you get there before I do, please prepare a gel or two and remove the plates from the 37c upstairs, thanks.
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*Team 2: Jay performed the minipreps on the 6 seedings.  She also set up the screening digests using the following quantities: 3 uL miniprep DNA, 0.53 uL XmnI, 0.33 PstI, 0.53 uL XbaI, 1.5 uL BSA, 1.5 uL Buffer2, 8 uL dH2O.  Jay ran gel; had very weak bands from our miniprep sample and nothing in the digested wells.
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Brock seeded 6 new colonies of E0422/C0012.  Prepared a restriction digest of R0062 and B0034 minipreps to screen.  Used the following quantities:  3 uL miniprep, 1.5 uL BSA, 1.5 uL Buffer2, .4 uL XmnI, .4 uL BanI, 4 uL H2O
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*Team 3: Jieun and Ashwini: First our gel screening from super concentrated (3 tubes of miniprep combined, protocol curtesy of Elvis (the king), THANK YOU ELVIS (thank yuh, thank yuh very much)!) luciferase gene had a very strong band around 4kb.(the uncut plasmid) The digestion of luciferase and pGFP (pGFP was provided by Elvis (blue suede shoes)) was successful and we ligated 10uL of luciferase insert with 2uL of GFP vector. The tube (lig GFP luc June 21/06) is stored in 4C fridge in the white rack. Please do not touch unless you are Elvis (all shook up)/Ashwin/Horia. Can't touch this . . .nanananananananana.
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'''June 22''' Thursday
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*Team 1: A gel was prepared and the digests from yesterday were ran on it. It was found that the sizes of the bands were completely weird and therefore it has been decided by the professor to redo the Topo ligation from the present digests this weekend. The plates for the Spanish DNA looked like a big lawn of bacteria.
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*Team 2: Controls from yesterday's seedings were cloudy, so they were disgarded.  Precipitated the DNA from yesterday's minipreps to concentrate it.  Loaded and ran gel of yesterday's digests.  The XmnI enzyme did not cut will but there were bands at ~650 and ~1400. One of the R0062 minipreps did not have DNA. Over the weekend Annette will seed 6 colonies for us of the C0012/E0422 ligation.
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*Team 3: Ashwin: Transformed 2 vials of Top10F cells, each with 10uL of pGFP-luc ligation. Plated 4 Petri dishes - 2 for each vial, one being 75uL and the other 150uL. Stored in 37C incubator at 5:15pm. Plates will be removed from the incubator tomorrow morning (i.e. after 16-18 hours of incubation) by Elvis, and stored in 4C fridge.
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*Grew and induced w/IPTG cultures of MJF465&367+pGFP+Eco-MscL. In a paradoxal outcome, uninduced cultures fluoresced bright green under the microscope, but induced ones showed no fluorescence. Mesmerized, we let it be and went for Grand Prix Weekend!
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'''June 23''' Friday
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*Team 3: Elvis seeded the ligation product from the plates from June 22. (Both plates showed fair amount of colonies.)
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'''June 24''' Saturday
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*Team 3: Elvis did the midiprep from the seeding from June 23.
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'''June 26''' Monday
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*The induced cultures showed some fluorescence this morning (RFU ~1500) therefore all is not lost. Seeded 5 tubes (5ml LB) with newly picked colonies from MJF465+pGFP+Eco-MscL 20uL plate. Let shaker-incubate overnight.
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*Team 2:  Miniprepped the 6 seedings from the weekend following protocol on May 9, 2006.  Set up screening digestion of E0422/C0012 ligation used the following quantities:  3 uL C0012/E0422, 1 uL BsrbI, 1.5 uL Buffer2, 9.5 uL dH2O.  Screening of gel did not contain DNA.  Transformed ligation into commercial Top10 cells.
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*Team 3: Jieun: The digest of ligation product had been prepared by Elvis, and was run on the gel twice (the first digest did not show any band, and the possible reason was the diluted status of midiprep prepared) but with some degree of frustration and disappointment, she had to leave the lab at 7pm. Earlier that day, Ashwin seeded new batch of ligation product.
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'''June 27''' Tuesday
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*Transferred 1mL of overnight cultures in 25mL LB and grew for a few hours. Elvis removed them from SI and placed in fridge for induction tomorrow.
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Team 1: Brainstormed about different ways to clone in order to get Fos-B and YFP on same plasmid and Jun-B and YFP on same plasmid. Did not come up with ideal strategy. Emailed resource for Fos-B and Jun-B DNA to get ideas. Decided to wait and see if they would respond and send us the sequences.
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*Team 2: Ligation plate from yesterday had many colonies.  Transferred to 4 deg. C.  Elvis seeded 6 colonies for us.
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*Team 3: The seeding from June 26 did not contain anything, therefore new seeding was done in the morning. Jieun seeded new batch of ligation product, this time with the pipette tips inside the tubes. She came back after 8 hours to see any sign of growth, but there was nothing. Elvis helped her seed another batch.
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'''June 28''' Wednesday
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*Cultures were incubated with different amounts of IPTG. O.D. and fluorescence were taken periodically.
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Team 1: Did 3 seedings, each one 2 microliters: Fos-B, Jun-B (Spain) and Fos-YFP-Jun-YFP (Michigan). Worked some more on cloning strategy. Came up with new idea. Going to PCR YFP gene from Fos-YFP-Jun-YFP plasmid and clone into the Fos-B and Jun-B plasmids from Spain. Designed primers and which enzymes with which we are going to cut our PCR product. Later on, mini-seedings were transferred to large 25 mL seedings by Jay. 
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*Team 3: It turned out there were some major seeding problems, and the possible cause would be the amp plate being unable to select amp-resistant colonies. Therefore, new plating was done (two plates, 75uL and 100uL) to be taken out from 37C the following morning.
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*Team 2: Nothing grew in the culture tubes from the seedings.  Poured new Amp plates.  Followed protocol on May 2, 2006.  Tranformed ligation into Top10 cells followed protocol on 6-26-06.
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'''June29''' Thursday
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*Graphs of O.D. vs time and O.D. vs Fluorescence were plotted. Probably only one culture contained the GFP, though, because of the sharper peak at 510. In order to test for MscL, hypoosmotic medium was prepared. Culture cells containing the plasmid and a control were brought to O.D. ~2.0 and 1mL vol. and shocked in 10mL hypoosmotic medium. The cells containing MscL reached O.D. 0.287 and the control reached 0.187.
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Team 1:Results: The Fos-B and Jun-B seedings grew but the Fos-YFP-Jun-YFP seedings did not grow. Tested primers on Primer Express to get right length for 60 degree Celsius temperature and to test primer-dimer action. Ordered primers for YFP PCR. Did midi-preps of the two seedings that worked. Isolated what we thing is a tube of Jun-B DNA and Fos-B DNA
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*Team 2:  Tranformation and plating from yesterday grew 7 nice colonies.  Colonies were seeded followed from May 9, 2006.
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*Team 3: There was technical difficulty regarding 37C incubator (aka it was not turned on), hence no colonies grew. The plates were kept in the 37C room, and were to be taken out the following day.
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'''July 1''' Saturday
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*Hmmm, I hope people haven't stopped working just because I'm at the north pole... here at the north pole, we have stopped working for a bit, due to bad weather, and are sitting around eating, getting fat, and sending email.  Quite the life.  There's snow.  Hope the ligations are working.
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'''July 3''' Monday
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Team 1: Decided to cut what was isolated from midi-preps w/ BamH1. Going to run digests on a gel to see if we get characteristic bands. Primers should arrive tomorrow for YFP PCR. Hope you're having fun Jay!
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*Team 2: Set up screening digestion of E0422/C0012 ligation made master mix with the following quantities: 3.75 uL enzyme BsrbI, 7.5 uL Buffer2, 48.75 uL dH2O.  This was divded into 5 tubes and 3 uL of miniprep DNA was added to each.  Ran a gel of the digested DNA and un-digested miniprep DNA and found that there was no DNA.  Did DNA precipitation following following protocol on 6/22/06.  Prepared screening digestion of the precipitated DNA.  Digested overnight.
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*Team 3: Ashwin and Jieun: New plating of the transformed Top 10F containing the ligation product was performed on two amp plates. For the future use, another ligation was performed using the same DNAs (Luc from June 21st, which had very strong bands on the gel analysis and GFP from Elvis) with the same ratio from June 21st.
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'''July 4''' Tuesday
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*Team 3: Jieun: Transformation of ligation product was performed (one vial of Top10F with 10uL of ligation product). The other 20uL of ligation product is stored in 4C fridge for overnight, and the plates containing trasformed cells are kept in 37C incubator room (starting from 5:30pm EST).
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*Seeded 2 colonies from pB10+Eco-Mscl in DH5a overnight.
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*Team 2:  Ran a gel of our precipitated DNA and digested DNA.  Found there was no DNA.  Seeded more colonies of the E0422/C0012 ligation.
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*Team 1: Did three seedings with 2 controls in 2 mL. Digested Fos-Beta a second time. Ran Fos-Beta on a gel but had no DNA in our lane. At 5 PM, added more LB to our seedings and left them overnight in incubator.
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'''July 5'''
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*Team 2:  Miniprepped seedings from yesterday, followed protocol on May 9, 2006.  Set up screening digestion of miniprepped DNA.  Used the same quantities as on July 3, 2006.  Ran a gel of this digested screening and undigested miniprep.  All 5 samples had DNA in them.  Unable to tell if the DNA was cut properly because the lanes were smeared significanly.  Made new TAE IX Buffer in an attempt to fix the constant smearing.
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*Team 1: Out of 3 seedings, only one grew. Did 3 mini-preps out of that one seeding. Ran 10 microliters of one miniprep in a gel to test if we got DNA. Nothing showed up. Decided to go back to culture plates. Transferred bacteria from one plate onto another amp plate to test if they are truly amp resistant. Put in 37 degee incubator overnight. Received primers for PCR! Will check plate tomorrow. If colonies grow, we're good. If not, need to consider redoing transformation.
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'''July 6'''
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*Team 1: We are good, we got colonies! Yay! Picked 10 off plate and did 10 seedings, each in 2 mL. Also did two digests, one of Fos-Beta DNA and one of miniprep from colonies off original pDsBiFC plate. Also just ran non-digested mini-prep DNA to test hypothesis if we are losing DNA with restriction digests. Ran a gel. The Fos-Beta showed up beautifully with the right size bands so now we know we have isolated both the Fos-Beta and Jun-Beta plasmids! However, no DNA showed up in the lanes for the miniprep. That's ok though because we have 10 new seedings. Came back in the evening to find that all 10 seedings grew and the controls were clear. Spun 10 tubes down and resuspended with P1. In the end, had two tubes of resuspended cells. Saved cells and culture tubes in fridge.
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*Team 2:
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'''July 10 Monday'''
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Team 1: Hi guys it's Adam, I'm just informng that I will not come Monday the 17th as well. Anyways all the best this week
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Latest revision as of 04:23, 12 May 2007

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May June July Team 1 May 2007

July 1 Saturday

  • Hmmm, I hope people haven't stopped working just because I'm at the north pole... here at the north pole, we have stopped working for a bit, due to bad weather, and are sitting around eating, getting fat, and sending email. Quite the life. There's snow. Hope the ligations are working.

July 3 Monday

  • Team 1: Decided to cut what was isolated from midi-preps w/ BamH1. Going to run digests on a gel to see if we get characteristic bands. Primers should arrive tomorrow for YFP PCR. Hope you're having fun Jay!
  • Team 2: Set up screening digestion of E0422/C0012 ligation made master mix with the following quantities: 3.75 uL enzyme BsrbI, 7.5 uL Buffer2, 48.75 uL dH2O. This was divded into 5 tubes and 3 uL of miniprep DNA was added to each. Ran a gel of the digested DNA and un-digested miniprep DNA and found that there was no DNA. Did DNA precipitation following following protocol on 6/22/06. Prepared screening digestion of the precipitated DNA. Digested overnight.
  • Team 3: Ashwin and Jieun: New plating of the transformed Top 10F containing the ligation product was performed on two amp plates. For the future use, another ligation was performed using the same DNAs (Luc from June 21st, which had very strong bands on the gel analysis and GFP from Elvis) with the same ratio from June 21st.

July 4 Tuesday

  • Team 3: Jieun: Transformation of ligation product was performed (one vial of Top10F with 10uL of ligation product). The other 20uL of ligation product is stored in 4C fridge for overnight, and the plates containing trasformed cells are kept in 37C incubator room (starting from 5:30pm EST).
  • Seeded 2 colonies from pB10+Eco-Mscl in DH5a overnight.
  • Team 2: Ran a gel of our precipitated DNA and digested DNA. Found there was no DNA. Seeded more colonies of the E0422/C0012 ligation.
  • Team 1: Did three seedings with 2 controls in 2 mL. Digested Fos-Beta a second time. Ran Fos-Beta on a gel but had no DNA in our lane. At 5 PM, added more LB to our seedings and left them overnight in incubator.

July 5

  • Team 2: Miniprepped seedings from yesterday, followed protocol on May 9, 2006. Set up screening digestion of miniprepped DNA. Used the same quantities as on July 3, 2006. Ran a gel of this digested screening and undigested miniprep. All 5 samples had DNA in them. Unable to tell if the DNA was cut properly because the lanes were smeared significanly. Made new TAE IX Buffer in an attempt to fix the constant smearing.
  • Team 1: Out of 3 seedings, only one grew. Did 3 mini-preps out of that one seeding. Ran 10 microliters of one miniprep in a gel to test if we got DNA. Nothing showed up. Decided to go back to culture plates. Transferred bacteria from one plate onto another amp plate to test if they are truly amp resistant. Put in 37 degee incubator overnight. Received primers for PCR! Will check plate tomorrow. If colonies grow, we're good. If not, need to consider redoing transformation.

July 6

  • Team 1: We are good, we got colonies! Yay! Picked 10 off plate and did 10 seedings, each in 2 mL. Also did two digests, one of Fos-Beta DNA and one of miniprep from colonies off original pDsBiFC plate. Also just ran non-digested mini-prep DNA to test hypothesis if we are losing DNA with restriction digests. Ran a gel. The Fos-Beta showed up beautifully with the right size bands so now we know we have isolated both the Fos-Beta and Jun-Beta plasmids! However, no DNA showed up in the lanes for the miniprep. That's ok though because we have 10 new seedings. Came back in the evening to find that all 10 seedings grew and the controls were clear. Spun 10 tubes down and resuspended with P1. In the end, had two tubes of resuspended cells. Saved cells and culture tubes in fridge.
  • Team 2: Did large scale digest for each of the 5 miniprep samples from yesterday. Expected bands at 1800 bp and 2030 bp. Ran gel of digests and had a band at 1800 and 500 bp. Came to the conclusion that the ligation product did not work properly. Screening for E0422 original plasmid minipreps: Cut with BsrBI.

July 7

  • Team 2: Ran gel of E0422 screening digests. Expected bands at 1800 and 1200 bp, well 1 had band at 1800, well 2 had band at 2000, and well 3 had band at 1800, 1600, and 1200 bp. Transformed 2 uL of E0422 brick supplied in homemade Top10 cells.
  • Team 1: Completed 2 minipreps from the cultures that grew last night. Then did 2 digests of miniprep DNA w/ EcoRI and BamHI and ran a gel of those digests in the afternoon. Purpose was to screen PLasmid DNA to make sure it is the pDsBiFC plasmid that we want.

July 9

  • Team 2: Seeded 4 colonies from transformation plate into LB + Amp for miniprep.

July 10 Monday

  • Team 1: Results from gel that was run on the 7th were not good because it seems the enzymes did not cut. Decided to run three more digests, 2 single ( 1 EcoRI and 1 BamHI, and 1 double w/ both of them) We then ran all digests on the same gel and we got our expected results! Yes! The only problem is the bands were so faint that you could barely see them.
  • Team 2: Miniprepped 5 mL of broth culture for each column. Prepared screening digests with BsrBI according to proportions listed on July 6/06. After running on gel, found only sample 4 showed a band, and it was at about 2000 bp. Annette recommended screening with a different enzyme (NotI). Set up screening digest with NotI. Run gel of digests with samples of today's miniprep to see if any DNA present in these minipreps. Well 4 was the only well to contain the correct DNA (band at 3000, 2000, and 1000 bp). Well 6 and 7 contained DNA but the identity could not be confirmed. Well 5 did not contain DNA, and wells 1, 2, and 3, did not contain the correct plasmid.

July 11

  • Team 2: Complete a large scale digest of the sample run in well 4. Cut using EcoRI, XbaI, and ScaI. Realized that this was very bad, because it destroyed our plasmid. Oops. Re-did the large scale digestion using EcoRI and XbaI, digested for 4 hours. Added 3 uL of CIP, incubated at 37C overnight.

July 12

  • Team 2: Performed DNA desalting to remove enzymes and small DNA fragment removed between EcoRI and XbaI sites. 53 uL sample volume -> 159 uL QX1, 3.8 ug DNA -> 10 uL QiaexII. Need to ligate C0012 brick (cut at EcoRI and SpeI) into E0422 with plasmid (cut at EcoRI and XbaI). Did calculations for ligation, set up ligation of E0422 and C0012, transformed into commercial Top10, and plated.

July 13

  • Team 2: Seeded 8 colonies from ligation plate for miniprep next day.

July 14

  • Team 2: Miniprepped 8 seedings, using 5 mL of broth culture for each miniprep. Screened the minipreps using Not I and Sca I, expecting bands at 2kb, 1500 bp, and 500 bp. Incubated for a couple of hours. When gel ran, observed bands at 4 kb, and a wide band at 3500 kb - perhaps the plasmid was only cut once, and the remaining uncut plasmid caused the wide band.

July 16

  • Team 2: Set up screening digests for 4 of July 14 minipreps, again using Not I and Sca I in case the problem was due to lack of incubation time. Incubated overnight.

July 17

  • Team 2: Gel ran of last night's digests showed no DNA, and smeary ladder. Jay suggested our minipreps may not be clean enough, so we washed them with Buffer PB. New screening digests with same enzymes, ran gel and found that there was no ladder shown! Replaced TAE buffer - maybe it's eating our DNA. New screening digest set up for overnight incubation with Ase I.

July 18

  • Team 2: Ran gel of last night's digests, expected bands at 1920 bp and 2200 bp. Observed band at 3000 bp. Set up another screening digest of all 8 minipreps from July 14, using Not I and Eco RV.
  • Team 3: Team 3 is making its triumphant return after a two week hiatus from the lab notebook!!! We first prepared a concentrated miniprep of the psp-luc+ by using 250uL of Buffer P1 to resuspend all 5 cell pellets. This miniprep was then digested with EcoRi and Hind 3 (digest labeled "psp-luc+mini prep conc 7/18"). The digested miniprep is placed in Cloning Fragments 6D and 6E. The "conc mini psp-luc+ 7/14" was also digested. The psp-luc+ and pGFP which had been digested yesterday was run on a gel. The gel was successful and the GFP vector band was seen at 3300bp while the luciferase gene band was seen at 1700 bp. The vector and the gene DNA was then extracted from the gel. The extracted DNA, the GFP vector "GFP 7/18" was placed in 6G and the luc gene "luc gene 7/18 was placed in 6F  :D

July 19

  • Team 3: The "luc mini 7/17" and "luc mini conc 7/14" digests were screened on gel, both yielding faint luc gene bands (1.7kb). The ligation of "luc gene 7/18" and "GFP (vector) 7/18" was performed: 15uL insert, 2uL vector, 2uL lig. buffer, 1 uL ligase. Incubated for 2 hrs at rm temp. 2 vials of Top10F' (1 commercial and 1 homemade) were transformed with the ligation, and plated.

July 20

  • Team 3: Yesterday's ligation plates were removed from 37C and placed at 4C (at 11:00). The plate with homemade transf. Top10F' had no colonies, but the 2 plates with commercial transf. Top10F' had medium-sized colonies. To be seeded at 16:00.

July 21

  • Team 3: A miniprep was prepared of the succesful seedings of the ligated GFP vector and the luciferase insert. The miniprep has now been digested to make sure that the DNA is in fact the ligated GFP and luciferase insert

July 25

  • Team 3: The digestion result revealed a few very important things. 2 minipreps out of 4 having been digested showed bands at 10kb and 8kb, and the other two at 4kb. The latter ones might contain the desired ligation product with the possibility of Sca1 inefficiency. The expected result from the digetion was 2300kb and 1900kb bands (adding up to approx 4kb, shockingly similar to the single bands that appeared on the gel). The minipreps of those latter ones are stored in the cloning fragment box (approx 30-50uL in each tube). A link to the paper about mechanotransduction activity in living cells has been posted under Journal Club Paper. Further analysis will be available after the group disccusion once Jieun snaps out of whatever she's going through.

July 25

  • Team 3: The 6 ligation minipreps (prepared yesterday) were digested with ScaI, and screened on gel. Again strange (very faint) bands appeared, this time above the DNA ladder. Possibilities are that these bands come from garbage DNA (contamination or chromosomal DNA), or ScaI isn't slicing properly! We should screen the minipreps to see if they actually any useful DNA, and try digesting with another enzyme.

July 27

  • Team 3: News To Horia: The Day Of Truth Has Arrived. According to Jieun "the truth is out there in the biohazard box." (long story- Jieun..was being herself = moody and Ashwini was being calm and rational) This time we digested with our best-buddies - EcoR1 and Hind III- and they did not dissappoint. The time had come, and the gel was placed on UV machine with Elvis' shaking hands. Jieun pressed the "Acquired" button, and voila. Did I say Voila? VOILA. BEAUTIFUL TWO SEPARATE BANDS PLACED WHERE THEY WERE SUPPOSED TO BE. We embraced this joyful moment by excessive hugging time and Jieun, despite her cranky and emotionally detached self, gave a big hug to her teammate,Ashwini. So, long story short. WE SUCCEEDED. BOOOOYAH. OHHHHH SNAPPPP!!!!

July 28

  • Team 2: WORKING LIGATION!!


July 29

Notes from Jay:

  • Team 1: Adam worked till late hours purifying the gel pieces, but didn't ligate because the ligase was locked upstairs and we're out.

Will anyone from Team 1 be in this weekend? I can do it if you like? Please email me if you're Team 1 and want to do something this weekend, I will be downtown this afternoon. To everyone, notes about the new Gel Purifcation Kit: please use ONLY ONE column for each piece that you cut out, no matter how much it weighs (try to minimize excess agarose). You can spin more than once. We only get 50 columns in the kit so please don't waste! Also, if you're purifying a piece with a molecular weight close to that of the loading dye (either the green or the purple), you will get some loading dye into your agarose and this will turn your melted piece greenish or purplish. This is OK and doesn't mean that you need to add sodium acetate. In fact, I have never once in many many many hundreds of these had to add sodium acetate. Always do the isopropanol addition, and the optional wash isn't necessary. C'est tout, bonne fin de semaine!

August 9

  • Team 3: To do the luciferase protein assay, we don't need to lyse the cells to react luciferase with luciferin. Jay found a procedure which can be found here: http://www.btci.org/k12/bft/bgt_background.html

Our present goal is to make LAR (luciferase assay reagent - 1 mM luciferin in 100 mM sodium citrate pH 5.5). We will make the luciferin and sodium citrate solutions separately. The overall plan is to prepare a seeding (in LB) of our ligation cells with O.D. of ~0.3, induce it with IPTG, centrifuge, dissolve the pellet (cells) in LAR, and thence observe the expected bioluminescent reaction.

August 10

  • Team 3: 500 mL of 100 mM sodium citrate (pH 5.5) was made yesterday and store at 4C. Today, we diluted a seeding of pGFP-luc cells (made on Aug. 8 by Ashwin) 1:150 - 600 uL seeding in 30 mL fresh 1X LB with 30 uL. Placed in shaking incubator until O.D. measured to be 0.365. 30 uL IPTG added to cells (1 uL IPTG/1 mL total volume), and placed in shaker for 2 hours. Also found that luciferin can enter E. coli without need of MscL channel ...

August 23

  • Team 3: The new plates with pGFP-Luc had somewhat less of a lawn than the previous ones. A miniprep screening digest was performed by Ashwin - with similar results as the previous screening done by Alida. It seems the bands of DNA move more slowly than the ladder, and the bands are thick and almost but not exactly in the right place. Please refer to the gel photo. New seedings have been done from the new plate.

August 24

  • Team 3: Beetle Luciferin is scheduled to arrive tomorrow (Friday). A seeding of ligation cells - diluted in fresh LB at an O.D. of 0.3-0.4 and stimulated with IPTG - must be spun down. Discard the supernatant, and the pellet of cells must be resuspended in 3-5 mL of the prepared sodium citrate. Transfer 1-1.5 mL of this solution to a microcentrifuge tube, and add ~0.25 mg of luciferin. The bioluminescence must be viewed in a dark room. The quantities described above (determined by Elvis and Ashwin) are estimated and may not be totally accurate.
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