Low PCR product - (Nov/14/2012 )

i am getting a low PCR product ! I have tried reducing the annealing temp, increasing the primer and DNA template conc. too. But still quite low. Anything I should try ?

-uday-

give details: how much total reaction; concentrations for everything; pcr program... we cannot help without these details

Andreea

-ascacioc-

Thanks for asking, Here are the details:
I use the Fermentas master mix according to supplier protocol.

DreamTaq Green PCR Master Mix (2X) 25 μl
Forward primer 0.1-1.0 μM
Reverse primer 0.1-1.0 μM
Template DNA 10 pg - 1 μg
Water, nuclease-free to 50 μl
Total volume 50 μl

PCR program
Initial denaturation 95 3 min 1 cycle
Denaturation 95C 30 s
Annealing 56C 30 s
Extension 72C 20 s
35 cycles
Final extension 72C 7min

Expected size of the PCR product is 253bp.

Hope this would help !

-uday-

how long is your insert? 20 s elongation is a bit too short for more than 1 kb

-ascacioc-

Hi!
If it is possible, you could try to reamplify your low PCR product making a nested PCR (I mean, using internal primers). This usually works very well.

-OA17-

Try adding 5% DMSO to the reaction (or a whole range from 1% to 10%). (doesn't help unless the problem is in the high GC content or secondary structures, but it's probably quicker just to try it, than analyse the sequence first).
0.5μM primers. 100 ng of template (I woudn't generally go higher than 300 ng of gDNA in reaction).
Tell me if it helped. DMSO saved few of my assays.

-Trof-

While you have mentioned that you have lowered your annealing temperature, how sure are you that 56 is the optimum temp for the PCR. have you tried anything lower? What are the Tm for your primers?

Just for additional info, what are you planning to do next, with the PCR product?
A gel picture of the low PCR yield would be of great help to all of us here.

-Ameya P-

uday on Wed Nov 14 21:32:53 2012 said:

i am getting a low PCR product ! I have tried reducing the annealing temp, increasing the primer and DNA template conc. too. But still quite low. Anything I should try ?

Hi,

There can be many contributing factors for getting a low PCR product. You can troubleshoot the following:

1) Check whether the primers you designed are perfect complementary to the DNA strands. Perhaps they are not specific which is one of the major reasons for low PCR product yield. Moreover they should not be self complementary or each others complement. If you designed them manually, I would suggest using a good primer design software. There are many tools available in the market. I personally prefer and use Primer Premier to design my primers and the tool has not disappointed me so far. Here is its link which I found on their website: http://www.premierbi...sign/index.html

2) You can try to reduce the volume of sample in the reaction mix if required and carry out another ethanol precipitation with the samples.

3) Try to optimize Mg2+ ion concentration and use a good proofreading enzyme with high fidelity.
All the best for your assays.

Cheers!!
Sabastian

-v.sabastian-