- I have changed the primer, taq, and even reextracted the DNA . Trying the temperature gradient didn't help. What confused me is that I can augment it before.
- I had suspected that there was something wrong with the primers, and buying them again didn't help. (Although another gene can be amplified)
Maybe you have an issue with self-complementarity of the primers, leading to concatenation of primers and/or products. If it is not possible to re-design primers, the addition of components affecting primer annealing like BSA, DMSO, etc., may help.
It may not have failed. A common mistake with nested pcr is to use too much template which leads to over amplifucation and the template anneals to itself and forms elongated concatamers of template.
The second round pcr should be done on 1ul of 1:100 dilution of the first pcr reaction mix ( this also dilutes the first primers) then only 15-25 cycles of second round pcr to get a clean second amplification. remember that every 10 cycles is 1000 times more pcr product, 20 cycles is 1000000 times more product so overamplification s very easy
I tried to electrophoreze the product from the first round and found that the first round was a failed amplification.
It may not have failed completely....just no visible amount of product but if there is any amount of first round product it should amplify well with nested pcr. Clearly something is amplifying to produce a smear. Try fewer cycles of the second pcr....maybe set up 4 pcr tubes and remove a tube at 15, 20,25 and 30 cycles
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