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7/26 PCR Troubleshooting continued
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Making & Running an Analytical Gel Electrophoresis
Previous attempts of PCR amplification of the YF1 and FixJ genes have shown signs of contamination in our PCR protocols. Specifically, there is the consistent appearance of a 1kb band in every sample in a previous experiment [LINK ENTRY], even in positive and negative controls. However, an experiment ran afterwards [LINK ENTRY] show no contamination. The cause of this contamination is still not understood.
This experiment tests different reagents used in Phusion PCR for contamination to see which one has been the cause of the undesired 1 kb band we have been getting in PCRs of YF1/FixJ.
PCR
Gel Analysis
Expected Results
If one of our reagents is contaminated, we should see the 1 kb band in every PCR except for the rxn that has the contaminated reagent swapped out.
In other words, if the H2O is contaminated for example, then PCR #1 above, using freshly autoclaved H2O, will not have the 1 kb band and should have the target amplicon. The rest of the rxns, still using the contaminated water, will have the 1 kb contaminant.
Notes
DNA Ladder type: (1kb plus NEB Quickload? etc.)
Positive control:
Negative control:
Gel 1
Start time:
Voltage:
Image of Gel 1:
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