From 2012.igem.org
Team:Technion/13_September_2012
Ilya
- Began working on adding our parts to the registry.
Inbal
- Restriction for BBa_B0015 (ter+pSB1AK3) with XbaI (3189bp).
- 2 starters of T3 RNAP with CM resistance (BBa_K346000).
Asaf
I figured that I don't have enough insert (Riboswitch with polymerase) for ligation with the Psb1c3.
I have decided to extract more insert from gel from the product I got from the fusion PCR with Tm of 55C.
I couldn't find the T7* product. I have already used both products of T7.
So I ran the Riboswitch with the K1F, T3 and N4 polymerase genes on an extraction gel.
I got the expected bands,but I didn't get the 1Kb pattern, and cut the 3.2 kb bands (as I expected from previuos gels).
I cleaned the DNA from the gel.
I cut all of my inserts (Riboswitch with different polymerases) with EcoRI and PstI.
I also helped Evgeni and made glycerol stocks of his starters,
and also centerfuge the starters so that he could miniprep them tomorrow.
Hila
- Last and final colony PCR for fragments 1C, 4C, 6C and 7C in the pSB1C3_MCS plasmid. No positive colonies were found.
- In addition I made a PCR reaction for fragments 1C, 4C, 6C, 7C just in case I won't find positive colonies. The PCR for fragments 1C and 6C didn't work again…
- Additional amplification to F4, F4* and F5. In addition today the 250bp overlap primer arrived, so I amplified fragment F4' which will help us in the Gibson assembly calibration.
Lior
Noa
Evgeni
Shahar
colony PCR for fragments 1C, 4C, 6C and 7C in the pSB1C3_MCS plasmid. No positive colonies were found.
Rachel
-Miniprep for pSB1AK3(BBa_B0015 biobrick) , the concentration was: 262.5 ng/ul.
-Digestion of pSB1AK3(BBa_B0015 biobrick) with XbaI, for 2 hours.
-Clean the SB1AK3(BBa_B0015) by column cleaning kit.
-I ran on gel the restriction products of SB1AK3(BBa_B0015) for 30 minutes, I also ran uncut plasmid as contro. The expected cut plasmid (linear) was 3300bp size. The uncut plasmid (pSB1AK3) ran up to 2200bp.
-The plasmid was treated with cip enzyme for 1 hour at 37C. Then it was cleaned bu column cleaning kit, the concentration was 62ng/ul.
-4 ligatins: T7*(K1F) RNAP + pSB1AK3, T7*(N4)RNAP + pSB1AK3, T7*(T3)RNAP + pSB1AK3 and control (pSB1AK3 without any insert). The ligation was at 16C overnight.
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