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Effects of more cDNA in PCR???


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#1 feri777

feri777

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Posted 10 August 2009 - 12:25 AM

Hi,

Standard PCR:

Taq = 0.5 Ál
10x CL buffer = 2.5 Ál
dNTP = 1 Ál
Primer = 2 Ál FWD & 2 Ál RVS
cDNA = 1 Ál
H2O = 16 Ál


OK, so my question is:

If I use 2 Ál of cDNA instead of 1 Ál, with 15 Ál of H2O what effects will this have on the PCR? The reason I am doing this is because my cDNA concentrations are relatively low. But if certain samples have higher cDNA concentrations will this negatively reflect in the PCR?

Not enough Taq, dNTPs for higher concentration resulting in lower PCR?

#2 autumn_lullaby

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Posted 10 August 2009 - 12:52 AM

Hi,

Standard PCR:

Taq = 0.5 Ál
10x CL buffer = 2.5 Ál
dNTP = 1 Ál
Primer = 2 Ál FWD & 2 Ál RVS
cDNA = 1 Ál
H2O = 16 Ál


OK, so my question is:

If I use 2 Ál of cDNA instead of 1 Ál, with 15 Ál of H2O what effects will this have on the PCR? The reason I am doing this is because my cDNA concentrations are relatively low. But if certain samples have higher cDNA concentrations will this negatively reflect in the PCR?

Not enough Taq, dNTPs for higher concentration resulting in lower PCR?


Hey Feri,

it's alright if you have more cDNA in your PCR reaction, especially if you think you have a low concentration of cDNA. You could get a bigger 'blob' of band and smears which indicate that you have a lot of product, that's all. 1 Ál of Taq is usually enough for 1 20 Ál reaction, you'll be wasting resources if you put too much Taq. And as for dNTPs, I think you won't get any products if you don't have enough of it because your dNTPs are the ones that put A, G,C, T on your strand.

Hope it helps =)




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