Lab Notebook

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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.