Team:Technion/22 August 2012

From 2012.igem.org



Team:Technion/22_August_2012
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Ilya

- Transformation of R0062+Fus1del in pSB1C3 and in pSB3C5 and of Fus2del in pSB1C3 and in pSB3C5. These clones will serve as positive controls for the plate reader assay. I think correct clones should give red colonies tomorrow.

Inbal

- 2 starters of the TOP10 bacteria containing the SP6 gene in pSB1C3 plasmid, I grow them for 8 hours in 37C incubator, then I mini preped the plasmidial DNA.
Also I did deletion PCR for the Bba_I13522 (the conc. of the template: 105ng/ul) , excluding the terminators in the part, I ran the PCR products on 1% agarose gel for 40 minutes. The expected band is 3kb size approximately. The results: A 4000bp band size (likely there was a mistake in the experiment).
After consultation with Hila, I decided to digest the plasmid (the original one, not the PCR-amplified) with Pst1 and then run it on a gel, so it will run in his linear form- The results wasn't comforting- there wasn't any band! I need to start all over again tomorrow...

Asaf

I ran the PCR products of the Riboswitch on extraction gel, and cut the bands from the gel.
I also purified the gel extracts.

Asaf and Shahar

PCR for extracting the different polymerase genes from the different plasmids.

Hila

- ligation for the MCS insert to the cut pSB1C3 plasmid.
- chemical transformation of the ligated product to Top 10 - Rb competent bacteria.
- recovery for pSB1C3+fuz_1 bacteria from glycerol stock.

please cross your fingers…


Lior

Great Success!! We got colonies! more ALP than xyIE.
We tried antother run for Chris's plasmids- this time with Tm of 62, 7 degrees more than last time.
And it worked! We got 4 chubby bands.
Colony PCR for ALP in order to verify the insert within the pT7 plasmid, we got 4 colonies out of 5 (4/5)

Noa

Evgeni

-Ligation of pPROLar cut with BamHI and HindIII with Bba_015. Reaction with no insert serves as control.
-Transformation of the ligated plasmid into TOP10 competent strain.
-Plating ligated plasmid+control on Kanamycin plates and growing at 37C overnight.


Rachel