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Team:Technion/10 September 2012
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Ilya
- Did colony PCR for the pCP+RS+mCherry and pCP+RBS+mCherry clones that Rachel prepared for me. There were some positive results but then I realized that they are useless. The previous insert in the plasmid was also mCherry, so I had no way of telling the difference between the background clones and my clones judging by colony PCR.
- Put starters of the colonies I picked for the colony PCR in order to do a test with BamHI digestion tomorrow to find the correct clones.
- Planned some aspects of the gibson attempts that we are going to do this week with Roee and Hila. Tomorrow we will try the assembly of fragments 4 and 5 with 100bp and 400 bp overlaps with different incubation times in 37 degrees followed by the incubation in 50 degrees which is instructed in the gibson protocol.
- Prepared a 0.6% gel for testing the gel electrophoresis conditions for the gibson results.
Inbal
- Digest pTetO+m-cherry with XbaI+SpeI.
-6 minipreps for T7* RNAP+ter+pSB1AK3: the concentrations are: 71,139,126,97,40.5 and 54 ng/ul.
-Digest T7* RNAP+ter+pSB1AK3 with XbaI (x6).lycerol stock for this part. I also did a g
-colony PCR for T7+ter+pSB1AK3 (2700bp) with 2 suitable primers (total of 6 colony PCR reactions)- after the PCR, I ran the products on gel, and 1 band was positive! I have a positive clone! yay! now I can continue with the cloning!
-I digested T3 native RNAP with SpeI and XbaI and cleaned it by kit, the concentration was 31.4ng/ul.
- I also cleaned the PCR pTetO+m-cherry product by kit and the concentration was 20.6 ng/ul.
-I cleaned the products of yesterday restriction (of T7+ter+pSB1AK3) and the concentrations were:11.8,10.8,6,12,12.2 and 17.8 ng/ul.
- Also, today I ligated between T3 RNAP and pSB1AK3 plasmid (1 hour, at 37C), transformed it into TOP10 bacteria and plated 100ul and rest.
-I did a restriction reaction of T7* RNAP+ter+pSB1AK3 with PstI enzyme (15 minutes, 37C),for verifying what I have. I ran the products on a gel, and compared it to an uncut plasmid (approximately 3200bp on the gel), and the experimental sample had a band in 6kb approximately. This is a good result!
-3 starters of T7*+ter+pSB1AK3 (AMP resistance).
-GOOD NIGHT!
Asaf
I ran the fusion PCR of the Riboswitch with the different polymerases on a gel. This time I got the required bands (3.2 kb)
and also 2.7kb and 500 bp bands.
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I ran the 56C Riboswitch+different polymerase on an extraction gel. I ran T7 of both 55 and 56C because I wasn't sure if he had a 3.2 kb band.
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Hila
- Colony PCR for five positive colonies from each fragment transformation.
- I found two positive colonies for fragment 2, one positive colony for fragment 3, and three positive colonies for fragment 8.
- In addition I started to create the Gibson assembly fragments. I started with F4 and F5. F4 was amplified using two different AS primers: one with 100bp overlap and one with 400bp overlap.
- Overnight ligation to fragments 1C and 6C in pSB1C3_MCS.
Lior
We decided to check the T7 promoter parts with bacterias with PET system. we transformed the T7 promoter to the BL21 bacteria.
We also made colony PCR for our Positive control plasmids (pTetO with ALP/xyIE)
Noa
Evgeni
- Due to continuous failure of Bba_015 insertion in pPROLar plasmid I'll try new strategy: instead of double digestion of pPROLar I'll do sequential digestion: HindIII-HF restriction -> Heat inactivation of HindIII-HF at 80C -> BamHI-HF restriction.
- In addition I'll do new Bba_015 restriction with BamHI+HindIII, maybe ligation problems were due to problem with biobrick restriction (Done by Rachel)
- Overnight ligation of newly prepared pPROLar and Bba_015
Shahar
-Purification PCR product of N4 RNAP (the one with pSB1A2 primers) by PCR cleaning kit. -Digest of N4 RNAP with XbaI and SpeI for creating sticky ends. -Clean the N4 RNAP after digestion by cleaning kit. -This insert (N4 RNAP) is ready for cloning.
Rachel
- Digestion Bba_0015 with HindIII-HF & BamHI-HF to creat sticky ends.
- clean the products after restriction. the concentration was: 19.8 ng/ul
