m******n
发帖数: 194 | 1 everything you mentioned is considered, good comments, esp the last one
for example, unknown SNP/mutation in your primer region will
certainly change PCR efficiency. Or DNA modification might change
primer binding. If you are a biologist, you would think of these first.
DNA 2nd structure is certainly another thing to consider, esp. since my
amplicons are short (~70-150 bp). My student spent quite some time on
this aspect (quite some $ on software as well) and results are dubious.
For DNA 2nd str | t**s
发帖数: 284 | 2 I have been working on splicing of a weak intron for some times.
When I tried to measure the portion of spliced mRNA by RT-PCR,
I actually observed similar phenomina as you got here.
In my assay, I place 2 primers around the intron (89bp) to run RT-PCR,
hoping that by measuring the ratio of the two PCR products (spliced
and unspliced) on agarose gel, I could get the efficiency of the splicing.
But we found that is not really true. When we compare PCR assay with
RNase Protection Assay, obviously
【在 m******n 的大作中提到】
: everything you mentioned is considered, good comments, esp the last one
: for example, unknown SNP/mutation in your primer region will
: certainly change PCR efficiency. Or DNA modification might change
: primer binding. If you are a biologist, you would think of these first.
: DNA 2nd structure is certainly another thing to consider, esp. since my
: amplicons are short (~70-150 bp). My student spent quite some time on
: this aspect (quite some $ on software as well) and results are dubious.
: For DNA 2nd str
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