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Team:Technion/28 August 2012
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Ilya
- Put starters of pSB1C3+Fus2, pSB1C3+Fus2del, pSB3C5+Fus2, pSB3C5+Fus2del, pSB1C3+MCS for the plate reader tomorrow.
Inbal
-I did a research on the SP6 gene that was sent to us from china- I found the sequence of the plasmid it came with, I also found the map+restriction sites of the plasmid (pACSP6R, or his other commercial name: pACYC184).
after that, I digested this plasmid with BamHI+HindIII and ran on 1% agarose gel. The expected bands were: 5600 and 350 bp approximately. The actual bands that I got were 3 light bands: 3kb, 2.5kb and 1.5kb and very light smear above them. Tomorrow I need to send for sequencing this plasmid with 2 primers (that I already have) to verify once and for good the sequence! I hope for the BEST!!!
- Transformation of BBa_K346000 (T3 RNAP) from yesterday to the TOP10 bacteria. I plated 100ul and 200ul on LB+Amp plates. The plates are in 37C incubator for the night.
Asaf
I ran a PCR reaction of the different plasmids that contains the polymerase genes (T7*,N4,T3,K1F,SP6)
in order to extract these genes.
Hila
Gel purification for fragments 2, 3, 4, 5, 7, 8 (with and without restriction sites).
Lior
Noa
Transformation of ALP + N4 T3 K1F Sp6, Xy + N4 T3 K1F Sp6.
Plating on CM plates.
Evgeni
- Made miniprep and restriction analysis from the new starters again. Still no positives. I will try to do ligation again, however this time I'll treat a pPROLar backbone with CIP to prevent self ligation, will raise insert:plasmid ratio to 5:1 instead of 3:1 in previous ligation and put ligation overnight instead one hour in 30 degrees.
- Digestion of pPROLar again with BamHI-HF and HindIII-HF + cleaning for the new ligation.
Shahar
Miniprep to pSB1C3+MCS(1) & pSB1C3+MCS(3)
