a******a
发帖数: 283 | 1 any trick to reduce the noise generated by the genomic DNA background? There
is no gene of interest in the genome, the primers only anneal to the viral
gene in the plasmid. I added the genomic DNA as background to generate
standard curve for future study (when measure genes instered in the genome).
However there is a lot of background when added the genomic DNA.
Primers work fine with only plasmids as DNA substrate. |
h******y
发帖数: 351 | 2 There may be many non-specific binding sites for your primers in genomic DNA
. Try to increase the annealing temp and do 3-step PCR instead of 2-step, o
r design better primers.
There
viral
).
【在 a******a 的大作中提到】
: any trick to reduce the noise generated by the genomic DNA background? There
: is no gene of interest in the genome, the primers only anneal to the viral
: gene in the plasmid. I added the genomic DNA as background to generate
: standard curve for future study (when measure genes instered in the genome).
: However there is a lot of background when added the genomic DNA.
: Primers work fine with only plasmids as DNA substrate.
|
G*****h
发帖数: 320 | 3 Design several pairs of primers and then run BLAST searches of these primers
against the host genome sequences (and closely related species) if they are
available.
Rule out these primers with high identities, particularly those with
relatively long identical sequences at 3' ends.
Make sure that 3' ends of your primers have at least one base (the more, the
better) that does not match any host genome sequences.
Test your primers using host genome DNA alone to ensure there are no non-
specific amplifications before applying to your regular experiments. |
a******a
发帖数: 283 | 4 Thanks a lot for all the input!!! |
