Hello, I’m beginner in doing PCR assay. So, the question is very basic. I did a pcr test and after runing the gel electrophoresis, I saw the target gene got amplified. I was told to use the DNA sample, which I could amplify the gene from that by pcr, as positive control and try another DNA sample for runing another pcr.
I think I made a huge mistake as it was my first time doing this test. I throw away the amplified pcr product! :(
If I run the same thing again can I have the same result? If so, what is positive control then ?
please help me with this question.
thank you.
If you replicate the PCR with the exact same conditions, the samples that resulted positive should again be positive, same goes for negatives. So, the sample that resulted positive in your first assay can be used as a positive control, and the samples that resulted negative can be used as negative controls. I am talking about the DNA samples not the PCR products. Using PCR product as positive control is, for me, a way of cheating and it is not informative as you wouldn't be using the same template (genomic DNA for samples vs PCR product for controls), so it wouldn't be an actual "control".
Hope it helps. I'd gladly answer any further related questions.
If you replicate the PCR with the exact same conditions, the samples that resulted positive should again be positive, same goes for negatives. So, the sample that resulted positive in your first assay can be used as a positive control, and the samples that resulted negative can be used as negative controls. I am talking about the DNA samples not the PCR products. Using PCR product as positive control is, for me, a way of cheating and it is not informative as you wouldn't be using the same template (genomic DNA for samples vs PCR product for controls), so it wouldn't be an actual "control".
Hope it helps. I'd gladly answer any further related questions.
Luis Antonio is right that the sample that worked will do well as a positive control that your pcr is working even when some of your other samples may not be working. You should also always run a negative control ( no dna in the tube but add everything else).
If this negative control evr shows an amplified band then your reagents or system is contaminated and you cannot trust your results until the contamination is cleared up
I agree with our colleagues. After you get the PCR product you can make sequence analysid to confirm your PCR condition. If you need to amplify PCR product you can do nested PCR.
- Badges
- Science method