pcr prob

[Sudheendra Rao N R]

Hi,
What is your reaction mix volume?
What is your desired pcr product size?
Primer dimers are more likely in 10 microliter pcr reaction mix.
Also they tend to be below 150bp.
Increase reaction volume to 25 microlitre atleast.
Calculate annealing temperature using wallace rule and use that (though it
is Td but it helps better for primers about 20 bp length).
Put primer only 0.2-0.4microM in total reaction mix.

i guess that will help.
Sudheendra.

On Sat, Feb 23, 2008 at 9:50 PM, harold b <theoharis from gmail.com> wrote:

> Hi, my negative control (no template with both primers and mastermix)
> consistently shows a stronger single band whereas my amplicons show a
> leading similar but weaker band followed by a fading smear reminiscent
> of non-amplified DNA. In each case the product with DNA is less
> intense. Can someone make sense of this?
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