Team:Slovenia/Notebook/June.html

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June 16th, 2009
We prepared competent DH5α cells and tested competence of DH5α and DB3.1 cells as follows: - DB3.1 with ccdB (incubation on Amp plate) - DH5α with p53 (incubation on Kan plate) - DH5α with PSBA (incubation on Amp plate) - DH5α with PSBK (incubation on Kan plate) We prepared 50 LB mini-prep flasks (10ml), LB plates with Kan, LB plates with Amp and 4×100ml LB media.

June 17th, 2009
We prepared our own competent DH5α cells. We inoculated transformed cells (from the previous day) into 10 mL of LB with antibiotic.

June 18th, 2009
We transformed competent cells DH5α with pLUC in order to test the competence. We isolated following vectors: PSBK_1, PSBK_2, p53_1, p53_2, PSBA_2, ccdB_1 and ccdB_2 and ran them (except vectors with ccdB domain) on agarose gel electrophoresis. Only p53_1 isolation was successful.

June 19th, 2009
pET3a and p53 vectors were cut using NdeI/BamHI. Three groups of competent DH5α cells were transformed with plasmids M13, pET3 and 7G and then incubated on LB Amp (M13) ad LB Kan plates (pET2 and 7G).

June 22nd, 2009
We separated the restriction products (pET3a and p53 vectors, that were cut with NdeI/BamHI) on gel electrophoresis, isolated them from gel using MINElute Extraction Kit and measured their concentrations. p53 had not been cut with both mentioned enzymes at the same time, but in a way that we got fragments d53 and dimtetra. We inoculated mini-prep flasks with colonies that had grown on Amp and Kan plates from 19th July.

June 23rd, 2009
We isolated M13, pET3 and 7G plasmids from over-night culture. We purified restriction products d53 and Dimtetra and ligated them into pETa (+ backligation).

June 24th, 2009
We transformed DH5α cells with the ligation products made on 23th June.

June 25th, 2009
Transformed DH5α cells were inoculated into mini-prep flasks.

June 26th, 2009
From transformed DH5α cells we isolated: - pET3a containing d53 - pET3a containing Dimtetra and performed control restriction. Gel electrophoresis of restriction products revealed that either restriction or ligation was not successful. Control restriction was done again, this time successfully. Therefore appropriate d53 and Dimtetra colonies were inoculated into 10 mL of LB+antibiotic in mini-prep flasks. Following primers were ordered: T7p-f ccdB-MCS-I-T7-p-r T7p-MCS-ccdB-f His-MCS-II-ccdB-reverse His-MCS-II-ccdB-r nYFP-r nYFP-f foldon-f foldon-r KSI-DP-f KSI-DP-r YFPc-f YFPc-r YFPn-mut-f YFPn-mut-r gyrB-f gyrB-r LL-37-f LL-37-r CutA1-f CutA1-r

June 29th, 2009
We isolated vectors containing d53 and Dimtetra. Gel electrophoresis showed that isolation was successful. BL21DE3 pLysS cells (for protein production) were transformed with the isolated vectors. Other group of BL21DE3 pLysS cells was inoculated on LB plates with chloramphenicol. We multiplied ccdB domain and p53 monomer with PCR using ccdB-f, ccdB-r, p53-f and p53-r primers.

June 30th, 2009
Gel electrophoresis of PCR products (see June 29th). BL21DE3 pLysS cells transformed with vectors, containing d53 and Dimtetra, were inoculated into 10 mL of LB+antibiotic.

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