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Problem with multiplex PCR - Troubleshooting (Sep/11/2010 )

Hi,
I am trying to setup a triplex PCR for M.tuberculosis
my product sizes are 123 bp, 162 bp and 590 bp
I am getting a faint band for the 590 bp product when i run my PCR
This is the details of the mastermix that i make
Reaction buffer(10x with 20mM MgCl2): 3 microL
dNTP`s 10mM : 1.5 microL
123bp primer Forward 10 micro M : 1.5 microL
123bp primer Reverse 10 micro M :1.5 microL
162bp primer Forward 10 micro M : 1.5 microL
162bp primer Reverse 10 micro M :1.5 microL
590bp primer Forward 10 micro M : 1.5 microL
590bp primer Reverse 10 micro M :1.5 microL
taq polymerase (5u/microL): 1.5 microL
DNA template : 5 microL
MilliQ water : 30 microL
My PCR conditions are
Initial denatuaration at 94 for 5min
denaturation at 94 for 1 min
annealing at 72 for 1 min
elongation for 1 min
final elongation at 72 for 10 minutes?

What can i do to increase the intensity of the faint band ??


And can i do a colony PCR with M.tuberculosis ?? i am asking this because it takes 3 days to extract DNA with the current protocol that i am using!! i have little time left before my next DC

-vinay118-

can you show your gel picture?

-adrian kohsf-

Advice 4 u:

1, adjust the final concentration of the primers for 590bp in rxn,because the 590bp is relatively longer than the others, the ampification efficiency may be lower than them correspondently.

2, a realtime PCR should be done to determine the amplification effciency of the 590bp primers. if the amplification efficiecy is low, plz re-design your primers.

3, check ur primers to ensure there are no dimers or hairpin esp, in the 3'end of the primers between them; Meanwhile, check the Tm value of these 3 pairs of primers. the same Tm value can make these 3 paires of primers to bind to the template in the same chance.

-Dicer-

Vinay,

To get a better band of that specific product size, increase the primer concentration associated with it. You could also lower the annealing temperature, but that might lead to increase in non-specific banding patterns in you gel.

-gt_ameya-

adrian kohsf on Sun Sep 12 08:31:31 2010 said:


can you show your gel picture?

will put up the picture in a day

-vinay118-