IGEM:UNAM LCG/2009/Notebook/Hydrobium etli/2011/09/17

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Band extraction

Abstract

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.

Details

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:

  • 90V, 95 min.

We didn't take any image of the gel, we just cut out the bands that we wanted.

Band Extraction

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.

Results

Helena Reyes ran a gel to see the results

Gel image

Lanes:

 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

Image:Aislado_de_banda_pbbrmcs-5,_hyda,_pfor.JPG

Gel 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).

Notes

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).


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