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Team:Paris Liliane Bettencourt/Notebook/2011/07/24/
From 2011.igem.org
Cyrille
Preparation of the biobrick K606003
The product of the PCR of the 22/07 was digested 5 min at 37°C using DpnI fast digest from Fermentas.
5 microL of the product nd the control where loaded on the gel, but no clear bands appears. The transformation was done whatsoever.
The aim here was also to test if he use of MH1 cells is mandatory or normal E. Coli cells works. S, the PCR product was transformed 2 times with the differents types of cells. A transformation positive control (S27 on the labbook reference) was also done.
The transformation was done by heat chock, and then plated. The bacterias are then grown overnight at 37°C.
Camille & Danyel
We first exrtacted from the overnight culture of S27 (cf Yesterday's page ) the PSB1C3 plasmid containing RFP.
We digested two of the miniprep with EcoR1 and Pst1 for 30mn in a waterbath at 37°C. We used Fastdigest enzymes.
We then run those two digestion product on a 1% gel and otained three bands : 1kb, 2kb and 3kb.
We extracted the 2kb band and purified it with a Qiagen kit.
In the same time we've run a gel with 1µL of the PCR product from last night PCR.
At this time, we were not very convinced of what we observed so to be sure to have something correct we launched again the same PCR as yesterday.
Discussion with the team the next morning show us that the results were ok.
