IGEM:UNAM LCG/2009/Notebook/Hydrobium etli/2011/09/18
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pBBR1MCS-5 extraction by PCR
I and Helena Reyes made a PCR from an extraction that she had made. The objective was to amplify and obtain the plasmid pBBR1MCS-5 without the RFP and its promoter in order to use the lasmid as vector to insert the anderson's promoter collection.
Apparently nothing was amplified, but it is curious that there are no bands neither for the lanes that had the supposed plasmid before the amplification, this means that probably the PCR did not fail, I think the problem was that we never introduced any template DNA (the plasmid) may be because the extraction failed.
I made 4 reactions, I had two different pBBR1MCS-5 samples, I made a 1/10 dilution for each. I ran a PCR for each sample diluted and not diluted.
I ran a gel to check out the result of the PCR and the final concentration of the products. Nothing appear in the gel.
1. pBBR1MCS-5 sample-3 no diluted (No PCR) 2. pBBR1MCS-5 sample-3 no diluted (PCR, tube 1) 3. pBBR1MCS-5 sample-3 1/10 diluted (PCR, tube 2) 4. ladder 5. pBBR1MCS-5 sample-4 no diluted (No PCR) 6. pBBR1MCS-5 sample-4 no diluted (PCR, tube 3) 7. pBBR1MCS-5 sample-4 no diluted (PCR, tube 4) 8. empty
There was no ammplification, the lines in the gel are not visible. It is strange that lanes 1 and 5 are also empty because this means that there is no plasmid in there, so probably we never put any template DNA into the PCR. Maybe the plasmid extraction did not work out.
The tubes are in Helena's red rack, the PCR ones and the plasmid ones too.