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Notebook: September 2011

Thursday, September 1 2011

Vincent PCR purified the pSB3K1-extended (no TetR) for use in the Gibsons and negative controls.

The Gibson assembly of J6-Plac-Lysis had produced a single colony. Vincent colony-PCRed it using two different sets of primers to amplify subportions of the lysis cassette. The first set of primers (30-f & 816-r) amplify an 800 bp piece whereas the second set (Seq-J6-RFP-f & Seq-J6-RFPlysis-r) amplifies a 1100 bp piece.

The gel seems to confirm that lysis is in the plasmid. We will send the plasmid for sequencing on Friday.

After taking a closer look at the plate from the platereader, it seems that none of the T7 variants did any lysing. This realization made it urgent that we discover whether or not the full T4-lysis cassette was really in these Gibson-assembled plasmids. The samples that had been sent in for sequencing had primers that seemed to amplify the backbone, instead of the lysis cassette. While this does not necessarily mean that the lysis is not there, it does suggest that all the PCRS and gels used to verify the presence of lysis were only showing the existence of the backbone.

Alina ran a PCR with different primers from Douglas' first attempts at putting together the 2700 bp cassette that amplify select regions of the cassette.

Friday, September 2 2011

Alina wanted to try a new extension PCR of the T4-lysis and a Gibson of the exteneded-lysis into the K1 backbone. The transformation was successful with tons of colonies. A colony PCR will be run on Monday.

Nadine came back in the lab and cotransformed pSB3K1 Pconst-TetR with J61002 Plac-RFP since the sequencing results were concluent, in order to perform IPTG and ATC platereader experiments. We had a lot of contaminated bottles, so she also made new SOC bottles.

Monday, September 5 2011

Alina ran a colony PCR on the K1-T7-lysis Gibson assembly.

After having set liquid cultures on Saturday, Nadine set up the platereader experiment as follows:

• J61002 Plac-RFP with increasing concentrations of IPTG
• pSB3K1 TetR-LacI + J61002 Plac-RFP with increasing concentrations of IPTG
• pSB3K1 TetR-LacI + J61002 Plac-RFP with increasing concentrations of ATC

She had also prepared co-transformations with pSB3K1 TetR-LacI + J61002 Plac-lysis, but the sequencing results indicated that the lysis cassette was wrong.