From 2012.igem.org
Team:Technion/21_August_2012
Ilya
- Glycerol stock of R0062+Fus1 in pSB1C3 and pSB3C5 and of Fus2 in pSB3C5.
- Restriction of R0062+Fus1 in pSB1C3 and pSB3C5 and of Fus2 in pSB1C3 and pSB3C5 PCR products with PacI.
- Gel purification of all restriction products.
- Ligation of all 4 reactions overnight in 16 degrees.
Inbal
-Returning on the PCR from yesterday, But with different Tm's with the same primers (for pSB1C3 & pPROLAR separately) . The results: still not successful- there weren't any bands closed to 2700 bp.
The conclusion- We (Shahar, Evgeni, Rachel and I) need to plan new primers for the RNAP's PCR.
Asaf
-I ran the PCR products from yesterday on gel agarose to check what I got, and I got the expected bands (500 bp).
-I planned the PCR reaction for extracting the different RNA polymerase genes for the different plasmids with the
appropriate overhangs (T7*,N4,T3,K1F,SP6,T7 w.t).
Hila
- restriction reaction with XbaI + PstI (HF) to plasmid pSB1C3.
- 1% gel for plasmid backbone.
today the gel look good!!!
gel purification for the MCS insert and the pSB1C3 cut backbone.
CIP for the plasmids back bone.
Lior
Today me and Noa did a second PCR run for Chris's plasmids- k1f, T3, pN4, sp6. only the k1f came out good. we'll change the Tm for better result in the future PCR programs.
we've done transformation for the xyIE and ALP in Top10 cells and plated them on CM plate.
Noa
Evgeni
Shahar
trying to do the PCR from yesterday again, But with different Tm's with the same primers (for pSB1C3 & pPROLAR separately) . The results: fail. there weren't any bands closed to 2700 bp.
conclusion- there are problems with the priners, and we need to plan new primers for the RNAP's PCR.
Rachel
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