IGEM:UNAM LCG/2009/Notebook/Hydrobium etli/2011/09/17
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I made with Helena Reyes an extraction from band of the plasmid pBBR1MCS-5 which I need to insert my promoters constructions in E. coli S17 and then conjugate with R. etli.
I had two different samples, at the end we ran a gel to see if the extraction was succesful, one of the samples worked out well and the other did not.
Now I have to measure the consentration of the sample that is Ok so I can calculates the ammounts I need for the ligations.
Helena Reyes made the extraction and digestion of the plasmid. We ran a low melting point (LMP) gel to choose the bands that we wanted. The gel details:
We didn't take any image of the gel, we just cut out the bands that we wanted.
We used the QIAGEN™ QIAquick Gel Extraction Kit, protocol. We wanted an increased DNA concentration so at the end we only added 30μL of the elution buffer as the protocol indicates.
Helena Reyes ran a gel to see the results
1. Fermentas 100-10000 ladder 2. pBBR1MCS-5 sample 1 3. pBBR1MCS-5 sample 2 The remaining lanes are not of interest for this analysis
It is clear that the sample 1 were succesful and the sample 2 were not. The band from sample 1 is between 4000 and 5000 which is good because the plasmid should be 4.7Kb, the concentration is not as much as the ladder main markers (60ng/0.5μL).
The tubes are in my rack (the one that has the digestions from the promoters).
With this sample I can continue to make the ligation (after determining the concentrations).