Lab Notebook

From 2006.igem.org

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May June

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:

July 10 Monday 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