|
|
| Line 2: |
Line 2: |
| | Content= | | Content= |
| | __NOTOC__ | | __NOTOC__ |
| - |
| |
| - | ===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)
| |
| - | <br>
| |
| - | <br>
| |
| - | We prepared 50 LB mini-prep flasks (10ml), LB plates with Kan, LB plates with Amp and 4×100ml LB media.
| |
| - | <br>
| |
| - | <br>
| |
| - | ===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.
| |
| - | <br>
| |
| - | <br>
| |
| - | ===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.
| |
| - | <br>
| |
| - | <br>
| |
| - | ===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).
| |
| - | <br>
| |
| - | <br>
| |
| - | ===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.
| |
| - | <br>
| |
| - | <br>
| |
| - | ===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).
| |
| - | <br>
| |
| - | <br>
| |
| - | ===June 24th, 2009===
| |
| - | We transformed DH5α cells with the ligation products made on 23th June.
| |
| - | <br>
| |
| - | <br>
| |
| - | ===June 25th, 2009===
| |
| - | Transformed DH5α cells were inoculated into mini-prep flasks.
| |
| - | <br>
| |
| - | <br>
| |
| - | ===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.
| |
| - | <br>
| |
| - | <br>
| |
| - | 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
| |
| - | <br>
| |
| - | <br>
| |
| - | ===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.<br>
| |
| - | Other group of BL21DE3 pLysS cells was inoculated on LB plates with chloramphenicol.
| |
| - | <br>
| |
| - | <br>
| |
| - | We multiplied ccdB domain and p53 monomer with PCR using ccdB-f, ccdB-r, p53-f and p53-r primers.
| |
| - | <br>
| |
| - | <br>
| |
| - | ===June 30th, 2009===
| |
| - | Gel electrophoresis of PCR products (see June 29th).<br>
| |
| - | BL21DE3 pLysS cells transformed with vectors, containing d53 and Dimtetra, were inoculated.
| |
| - |
| |
| - | }}
| |
"
