I have only been able to amplify a smaller segment (~200bp) of a viral nucleoprotein gene. i have increased my extension time and it still is coming out negative.
The template seems to be rich in hairpin structures. To overcome this problem, use 5% DMF in the PCR reaction and decrease the annealing temperature by 10-20°C.
Hope this helps.
Jochen
As suggested by Jochen, you can use 5% DMF or DMSO in the PCR mix.
If your primers are for the 1.8 kb amplicon, then getting a 200bp fragment is surprising!!
Could be due to non-specific binding of primers, in that case, decreasing annealing temperature wont help. I would suggest to standardize your annealing temperature 5 to 2 degree below the melting temperature of the primers. Even before trying the above, check your primers using BLAST against the viral genome sequence for the specificity of your primers.
And one more this, what is the source of your template? is it the proviral DNA (plasmid carrying pro-viral DNA) as template or the virus infected cell genomic DNA as template?
Hope this helps!!!
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