after getting normal pcr product till about 2 months ago, when i started my work again. the PCR started showing light bands, dark smear a little away from the bands and prominent dimers. the PCR kit was new. conditions were the same as before. i guess it could be the quality of the dna but with 'bad' dna i shouldn't get any bands right? ideas? should i optimize conditions again?
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#2
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Posted 18 August 2009 - 08:32 AM
TaniaShakoori, on Aug 18 2009, 08:24 AM, said:
after getting normal pcr product till about 2 months ago, when i started my work again. the PCR started showing light bands, dark smear a little away from the bands and prominent dimers. the PCR kit was new. conditions were the same as before. i guess it could be the quality of the dna but with 'bad' dna i shouldn't get any bands right? ideas? should i optimize conditions again?
Rather than resetting the optimized PCR conditions, I would..
(1) Check it on old DNA template
(2) Check it on a fresh DNA template
(3) Check whether the primers had been stored right during this period. Otherwise order new.
(4) Use all fresh reagents for PCR.
(5) Make sure that the cycler works well with other PCRs.
If these don't work, then only I would change the PCR conditions such as temp, time and MgCl2 etc.
#3
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Posted 21 August 2009 - 09:10 PM
cellcounter, on Aug 18 2009, 09:32 AM, said:
TaniaShakoori, on Aug 18 2009, 08:24 AM, said:
after getting normal pcr product till about 2 months ago, when i started my work again. the PCR started showing light bands, dark smear a little away from the bands and prominent dimers. the PCR kit was new. conditions were the same as before. i guess it could be the quality of the dna but with 'bad' dna i shouldn't get any bands right? ideas? should i optimize conditions again?
Rather than resetting the optimized PCR conditions, I would..
(1) Check it on old DNA template
(2) Check it on a fresh DNA template
(3) Check whether the primers had been stored right during this period. Otherwise order new.
(4) Use all fresh reagents for PCR.
(5) Make sure that the cycler works well with other PCRs.
If these don't work, then only I would change the PCR conditions such as temp, time and MgCl2 etc.
thanx i'll try that
