I performed PCR yesterday afternoon and let it run over night on setting at 6 C in the thermocycler. This morning I prepared my 2% agarose gel and after it set, I placed it in the electrophoresis chamber and loaded 10ul of the samples into the wells. The volts used were 104-105v for about an hour and a half. When I took it to the imager, I got these faint bands showing. I just would like to know where in the process did I go wrong.
If your negative control is clean, you can't say if there's a contamination, but double bands are often due to non specific primers binding. If you're using specific primers, try to increase the annealing temperature to get the correct band size.
Its definitely not due contamination or else due to band elusion. if it is then band would be smeared out. I would recommend Aissa Saidi Suggestion. if your using genic SSR primer then.
1. Increase annealing temperature up to +3 Celsius. (or else go for Gradient PCR)
2. increases PCR amplifications up to 35 cycles in order to increase Band intensity.
3. Check the GC concentration (GC%) of primers .
Run an assay with 2 samples which have no dna in them and one normal dna sample.If the negative controls amplify bands of the right size then you have contamination
You can not say these band a contamination until you run a negative control and a positive control with the sample pcr product. I would suggest do electrophoresis again with positive and negative control to decide of there is a contamination or not. On the other hand if you still get these bands change the anealing temperature. Mostly, the wrong anealing temperature can give you multiple bands so make conditions little stringent to avoid non specific primer binding. You can also check if your primers are not binding multiple sites doing inslico PCR.
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