From 2012.igem.org
Team:Technion/22_September_2012
Ilya
- Wiki, wiki, wiki...
Inbal
-cip reaction for pSB1C3: 1 hour at 37C. After cleaning the Concentration was 45ng/ul.
-miniprep for all the starters I did yesterday...
-I checked the plasmids after miniprep by restriction enzymes- then, ran the products on gel and I have only 1 positive result with T7*RNAP+BBa_B0015 and T3+pSB1AK3- I continued working with those colonies to eventually ligate between pTetO+mCherry to the backbones and then transformation to TOP10 bacteria.
Also, I ligated pTetO+mCherry to pSB1C3 backbone and transformed it to TOP10 cells.
The transformed cells were incubated at 37C incubator overnight.
Asaf
-I have made glycerol stocks of all my starters (the selected clones from my long RS+polymerases transformed bacteria).
-I minipreped the starters and got very high concentrations.
-I did a PCR reaction for all the minipreped parts to add prefix and suffix to just the RS+different polymerase
(I have cut the rest of puc19 remeains from the insert).The PCR progrem was for Tm of 56,56.7,57.7 C
-I ran the PCR products on gel and got several positive results and several negative ones. I got enough positive
results for every polymerase type. The required band was 2919 bp.
-I have desided to continue to restriction with the following products: K1F-2, N4-1,N4-2, T7-1, T7-2, T3-3, T3-4.
-I cleaned the selected products and got a very high concetration (244-954 ng/ul).
-I cut the selected products with EcoRI and PstI restriction enzymes.
-I cleaned the products and got lower concentration (22-108 ng/ul).
-I ligated the different products to Psb1c3 plasmid that had been cut previewsly with EcoRI and PstI and afterwards
went to SIP reaction by Rachel. I didn't have left enough plasmid for the negative control.
-I transformed the lygated Psb1c3 and RS+poly to Top10 E.coli strain.
-I went to sleep.
Hila
- Colony PCR for positive clones from the transformation.
Unfortunately no positive clones yet to be found…
- Gel purification for all fragments with 100bp fragments overlap.
Lior
Noa
Evgeni
Shahar
Colony PCR for positive clones from the transformation - no positive clones yet to be found
Gel purification for all fragments with 100bp fragments overlap.
Rachel
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