Muliplex PCR problems!!
junk at hgmp.mrc.ac.uk
Sat Apr 20 05:06:58 EST 2002
Historians believe that in newspost
<Ghaw8.987$On1.2786360 at newsserver.ip.pt> on Sat, 20 Apr 2002, Ricardo
Dias <rpmdias at clix.pt> penned the following literary masterpiece:
>I use the same
>0,3mM of each primer and all the same conditions from individual pcrs) some
>pairs work well with all strains, some not.
>and some pairs don't amplify nothing at all.
>Any one have experienced this kind of problems or have some advice ??
That is not surprising at all.
For starters you need to make sure that the primers do not primer
against one another.
You need all primers to have identical Tm's.
You need a hot start enzyme.
You will have to optimise primer pair ratios so that one pair does not
out compete another pair and do that for all 5 pairs, adjusting each
until you get what you want.
Expect to spend a lot of time and even then you may not succeed :-((
I love deadlines. I especially like the whooshing noise they make as
they go flying by.
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