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nested PCR with low target DNA?


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#1 Mozzie_man

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Posted 20 July 2011 - 11:51 PM

Hi guys,

I was hoping to hear any ideas you may have to help me solve my PCR dramas. I am trying to amplify some insect DNA (extracted using a commercial kit) with insect-specific primers (within the "barcoding" region of COI). The product size is around 150bp.

Unfortunately, I can't seem to get any visible products unless I use a nested PCR in which I initially amplify a 658bp region of COI and then further amplify 1uL of that product with the insect-specific primers.

I have tried various combinations of annealing temperatures and template volumes (even diluting templates 1:10, 1:100, 1:1000), played around with MgCl2 concentrations, but still no luck getting the insect-specific PCR working on its own. All I get is primer-dimers.

So for some reason, once the target DNA is in higher concentrations (i.e. after being amplified in an intial PCR), the cycling parameters work really well.

My PCR recipe is listed below, along with the cycling parameters:

F (5uM) = 1uL
R (5uM) = 1uL
MgCl2 (50mM) = 0.4uL
dNTPs (10mM) = 0.2uL
Buffer (10x) = 1uL
Biotaq (5u/uL) = 0.05uL
Template = 1uL
SMQ = 5.35
Total = 10uL

94*C - 3 min

16 cycles (annealing temp decreases by 0.5*C/cycle):
94*C - 30 sec
61*C - 30sec
72*C - 30sec

34 cycles (annealing temp stays at 53*C for all remaining cycles):
94*C - 30 sec
53*C - 30 sec
72*C - 30 sec

72*C - 10 min

I am trying to investigate the diet of an insectivore and need a short product as DNA in fecal material will be degraded. However, I still need to get my + control (insect DNA) working before I can move on to the more difficult samples.

Any ideas or suggestions that you may have will be greatly appreciated!

Cheers,

Mozzie_man

#2 Trof

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Posted 21 July 2011 - 08:49 AM

Hope I'm not missing something in your post, but if your product is amplified in nested PCR only, the obvious reason would be that you just have to little of your template to be visible after first rounds of PCR. Nested-PCR is a valid method and is used for amplification of scarce product like tumor-specific translocations in DNA of bunch of normal cells. You just need to take great care of contamination in your reactions as the danger of it is multiplied.

But nested PCR usually looks like undetectable product in first PCR, then diluting the product (even if it's not visible) and running second round. You don't mention if your 658 bp product is visible after the first PCR or not. If you see a 658 bp product and not the smaller one within this region that could mean either (1) not all 658bp products have your insect-specific target inside for some reason or (2) your insect-specific primers are not efficient (strong dimer formation, mismatch) enough to create a visible product, and only amplify it when there is a huge excess of target template.

Also I'm bit lost in your PCR program, those 16 and 34 cycles are both in the first round of PCR or the second program is for the nested? Because if you run 16+34 in one PCR round (that means another 50 for nested) that is way too much.

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#3 epimaster

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Posted 01 August 2011 - 07:43 AM

Hi Guys, I wanna do long PCR around 2Kb PCR product but I dont know which polymerase I can use, I know Pfu works well but there are some other options? Thank you for any reply :)




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