From 2012.igem.org
Team:Technion/29_August_2012
Ilya
- Glycerol stock of pSB1C3+Fus2del (freezer position G2) and pSB3C5+Fus2del (freezer position G3).
- Plate reader assay for pSB1C3+Fus2 and pSB3C5+Fus2 with the positive (pSB1C3+Fus2del and pSB3C5+Fus2del) and negative (pSB1C3+MCS) controls.
Inbal
Transformation for pACSP6R and for BBa_K346000 (native T3 RNAP) to TOP10 bacteria, the resistances were Tetracycline and kanamycin respectively. Then, I plated on plates and incubate them in 37C incubator overnight.
Also, I did 2 PCR reactions for T7* RNAP that we got from Chris, each reaction with different primers ( one primer compatible for the pSB1A2 plasmid, the second is compatible for pPROLAR plasmid). The Tm was 58C, according to Roee's guidance.
After the PCR, I ran 5ul in 1% agarose gel, and I saw a desired 2700bp sized band with the pPROLAR primers, but no bands in the pSB1A2 primers. I don't know what else to do :/
I digested the pACSP6R plasmid with BamHI-HF enzyme- the results: good :] - after running it on gel, I expected a 5.9kb band, and that's what I saw in the gel! finally an appropriate result!!!
Asaf
-I checked the results of yesterday's PCR for extracting the polymerase genes on gel agarose,
and got two bands in T7*,T3 and K1f: one in 2kb and one near 3kb, and no bands in SP6 and N4.
I realized that I put an incorrect elongation time and this can explain the double band.
The Sp6 gene was probably defected.
-I ran another PCR reaction for the polymerase genes. This time with the right elongation time, but with Tm of 60C.
I didn't got any bands with the exception of T3.
Hila
Lior
Noa
Colony PCR for ALP bacteria described yesterday.
Positive results for: ALP+ T3 , N4.
Evgeni
- Overnight ligation of pPROLar backbone with Bba_015
Rachel & Shahar
PCR to Chris's Rnap with the new primers. The PCR with plac/ARA primer and T3, K1F & T7* templates, were successful.
The PCR with A@ primer and T3, K1F, N4 & T7* templates, failed.
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