Lab Notebook

From 2006.igem.org

Revision as of 03:59, 28 June 2006

Home

May 15: Transformed Top10F' bacteria with the pGFP vector containing the Luciferase gene (hopefully the ligation worked this time).17:22, 15 May 2006 (EDT)

May 16: Transformation of Top10F did not work. The cause was an error in the extraction of the DNA from the the agarose gel. Completed a new agarose gel extraction of pGFP plasmid DNA, as cut by EcoRI and HindIII. Prepared a new seed of Top10F bacteria transformed with luciferase. Prepared a midiprep seed of EcoMscL.

May 17: The control of the miniprep? of luciferase was murky. This could have been due to contaminants in the LB, water, ampicillin, or pipet tips. A miniprep of both luciferase and EcoMscL was conducted. A new seed of luciferase was prepared. The miniprep of luciferase was digested with EcoRI and HindIII. The miniprep of EcoMscL was digested with XmnI. A gel with the digests was run but the EcoMscL showed no bands and the luciferase only had faint bands.

May 18: The controls of the seed of luciferase were clear. A miniprep of the new luciferase seed and the old EcoMscL seed was conducted. The same restriction enzymes were used to digest the plasmids. A gel was run with the digests. There were two faint bands on the gel with the EcoMscL when there should have been three. Thus a new batch of Stable 3 bacteria were transformed with EcoMscL through heat shock and plated. The gel with the psp-Luciferase turned out well and the gene was extracted from the gel. Ligation was not completed since the microcentrifuge tube with the pGFP cut vector could not be found due to labelling issues.

May 19: The Stable3 plates were good. We Seeded with 5 microliters of Amp in 2.5mL of LB and 2.5 mL of sterile water and placed in shaker-incubator overnight. We also ligated pGFP & Luciferase (+2 controls, c.f. lab manual) incubating at room temp for 1h. The ligation was then put in the freezer.

May 21: Put up an example of cloning for oscillator.

May 23: A transformation of the luc pGFP ligation product was performed. It was reasoned not to do transfomation on the control (with no ligase) in order to save Top Ten colonies. Also only the transformation of only the ligation with 12 microliters luciferase and 3 microliters pGFP was done, not the 6 microliter luciferase one for the same reason as mentioned above. Only half (10 microliters) of the 12 uL-ligation was used, the rest stored in freezer. The result was plated. Regarding the MSCl, the seeding from the 19th did not work, both the controls were found to be murky (ampicillin and LB as well as LB only) therefore this was performed again.

May 24: 4 or 5 colonies on plates. Seeded 3 colonies in 3 tubes with 5mL LB. [Initially believed there were no colonies and ligation didn't work. We ran a gel of the ligation to see if it contained the Luciferase gene or any DNA at all (since the extraction procedure might not have worked).] It turned out that the Gel was actually fine. There was DNA in the Gel that corresponded to the luciferase insert and the pGFP plasmid. The recombinant plasmid was not seen, but it was not necessary to see it because usually only a small amount of recombination occus. So the plates were re-examined and it was found that on the 150ul plate there were indeed some faint colonies. These colonies were then seeded, 3 colonies and 2 controls picked. Miniprep of Stable 3 bacteria with pB10 vector containing MscL was performed.

May 25: The seeding did not work. It was done again, leaving the pipet tip in the tube. The miniprep product (pB10-MscL) was digested with Xmn1 and gel was run, to check the identity of the DNA. 3 new ligations of pGFP-Luciferase were performed: 1uL-vector/15uL-insert ; 1,32/4,43 ; 4/12. -15:00, 25 May 2006 (EDT)The Gel Showed absolutely nothing, the laddder was smeared and there was no DNA in the lane.

May 26: It was found that there was nothing in the seeding, as well the new ligations/transformations did not work. Worked on midi-prep of MSCL and digested luciferase and GFP with same enzymes: EcoRI and Hind III. Ran digests on a gel and got two very faint bands in wells 5, 6 and 7 for luciferase plasmid and gene but no bands in wells 2, 3 and 4 for GFP. Cut out luciferase gene and put gel in two tubes in freezer labeled "luc insert" with the date. WE think the reason there was no GFP is because we may have cut the insert instead of the plasmid because we used GFP DNA from May 16th, DNA which could not be identified as to whether it was the whole plasmid or just the GFP gene. Decided to make a square table to increase organization and to avoid confusion. Adopted new labeling technique where paper boxes correspond to actual boxes in freezer. Everytime we put something in the freezer, we indicate on paper in the appropriate box what we put there (name of plasmid), when (date) and who did it (first name). Now a message from Horia about tomorrow's fundraising party: Hey guys I just want to say that I won't be able to come in today, I still have to run some errands for the party tomorrow. Ashwin, can you bring my notebook tomorrow? And Ashwini, are you still interested in crafting a donation box? And who wanted to make snacks/brownies/cookies again? Just let me know by email or phone, so i get this organized - in my head at least:) Horia 12:07, 26 May 2006 (EDT)

May 29:Goals for this week: produce and screen midipreps of pGFP and EcoMscL. Grow a batch of chemically competent BL21 (mscl-) E. coli. Prepare minimal medium and thin imaging coverslips. Transfect cells with pGFP and image/photograph the fluorescence (can we see them moving? Do we need a video camera?) Organize plasmids and cloning fragments. Determine schedules and communication strategy for cloning. Belinda and Juilia prepared minimal medium solutions. Aaron and Brock ran a gel on the digest for MSCL but there was no DNA seen. The Bio bricks are finally in!!!! Ashwin and Adam did midiprep of pGFP and got a nice pellet. Also did 2 small-scale seedings of MscL, one from Stable3 cells and the other from DH5a.

May 30: We will have an ORGANISATION MEETING at 12pm in the B floor conference room. Coffee, tea and cookies will be served. PLEASE be there, because we need everyone to help in keeping things organised. If you REALLY can't make it, I will send you minutes of the meeting, but it is critical to have everyone start out on the same wavelength.
EcoMscL seedings were good. Carried out 2 minipreps: seeding from DH5a (Ashwin, Adam, Alex) and Stable3 (Aaron, Jieun, Brock). Digested the Eco-MscL minipreps with XmnI and ran a gel. The bands are faint but at the right position (3888 & 1420). NB: for screening use 2-5uL of miniprep and a total of 15uL solution (we used 50uL => faint bands). Stable3 bands looked better than DH5a.

May 31: Julia and Adam met in the morning to discuss Team 1's project w/ Jay. Decided to focus on Fos and Jun plasmids. First experiment will be to try to transform those two plasmids together into the same bacterium. If Alex gets this message, he can go ahead and start this experiment. Alex, ask Jay for the two plasmids and then all it is just a transformation. Teams 2 and 3 did research on their individual projects. Designated a binder for each team's plasmids, papers, etc. Had a presentation on iGEM from Andrew and talked about some good project ideas and fundraising ideas.

• Team 3: Today Ashwini, Ashwin and Jieun did background research on luciferase and luciferin in the morning. We found out that luciferase (firefly luciferase, the plasmid vector available in the lab, and this is a monomeric protein unlike the bacterial luciferase that is a heterodimer) is too big for MscL channel. (The dimensions were approx 119.53 x 119.53 x 94.68 Angstrom while the maxium diameter of MscL is only 40 Angstrom.) Making luciferin getting into the cell through MscL was considered, yet this would be very inefficient since it is applied in the opposite direction of the mechanism of openig MscL channel. Instead, we decided to take on from the following idea: while luciferase is being expressed in the cell, we can change the environment (ie.pH change causes different colour emissions of luciferase) or we can allow small molecules to go through the cell membrane and work as a switch, per se. We were also interested in Elvis' suggestion from yesterday's meeting when he talked about controlling bacterial growth to formulate a "picture." (By combining the changing of colours and controlled shape of bacterial lawn, we could create a domino effect where the cells would light up/change colour due to the adjacent cell activities.) We also found out that the presence of luciferin is crucial for luciferase activity since it provides ATP catalysis and O2. As of now, we are still at finding related articles and information. - Jieun (12:14am June 01/06)

June 1:

• 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
  Some interesting websites to revisit http://www.jbc.org/cgi/content/abstract/275/9/6047
  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§

• 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.

• 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.

• 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

• Team 2: Dissolved the BBa_I5610 in 10 uL 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 uL on one half and 20 uL on the other half.

June 2:

• 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. Update: 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.

• 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.

• 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.

• 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".

• 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.

NB: Two dH2O bottles smelled like Romanian toilets, so I threw them out and placed the remaining bottles under the pcr enclosure.

June 4 evening:

• Elvis (the king) seeded MJF465-pGFP cells in 1mL LB with AMP

June 5:

• 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).

• 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.
• bJunYN155
• Forward: 5'-CACCGTGTACGGTGGGAGGTA-3'
• Backward: 5'-ACTGGGGAGGGTCACAG-3'

• bFosYC155
• Forward: 5'-CACCTTCTAGGCCTGTACGGAAGTG-3'
• Backward: 5'-GACCATGATTACGCCAAGCTA-3'

• 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.

• 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.

June 6

• Team 1: Absolutely nothing was done today as our primers did not arrive yet.

• 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.

• 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).

• The induced MJF465-pGFP showed happy bacteria swimming but absolutely no expression of GFP under the uber microscope in Jay (z)'s lab.

• Elvis (the king) plated 40uL of the culture on chloroamphenicol plates to verify its identity.

June 7

• 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.

PCR (by Adam):

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.

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.

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.

• A: 80 uL PCR water
• B: 10 uL PCR buffer
• C: 4 uL dNTP's
• D: 2 uL each Forward and back Primer
• E: 1 uL BSA
• 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.
• G: 2 uL DNA, the Diluted solution

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.

• 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.

• 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.

• 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.

• Alex prepared 2 gels for our PCR products tomorrow.

June 8 Thursday

• 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.

• 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.

• 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.

June 9 Friday

• 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.

• Team 2: Transformed Top 10 with B0034. Seeded I15016 and C0012.

• 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).

• 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.

June 12 Monday

• 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.

• 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.

• 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.

• 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.

June 13 Tuesday

• 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.

• 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.

• 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!

• 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.

June 14 Wednesday

• 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.

• 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.

• 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.

• Elvis (the king) has left the building. Until Friday.

June 15 Thursday

• 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.

June 16 Friday

• Team 1: Massive minipreps from the seedings were done 5 for Fos and 5 for Jun

June 19 Monday

• 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.

• 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.

June 20 Tuesday

• 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.

June 21 Wednesday

• 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.

• Team 3: 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.

June 22 Thursday

• 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.

• Team 3: 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.

• 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!

June 26 Monday

• 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.

June 27 Tuesday

• 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.