PCR on PCR product

[danfive]

Originally Posted by jiajia1987

I am sorry to add to this, but I do not get why the concentration of the PCR product in the reaction mixture would be a problem. If the primer used in the first PCR anneals to the template and makes a PCR product, the PCR product should be similar to the template, with the ends similar to the primers used in the first PCR, right? Since that is the case, why would the second PCR be a problem? I am confused.

Well the first PCR makes millions of copies (maybe tens of millions). These copies will 1) Bind up Mg++, so your optimized PCR is now messed up because of the ratio of Mg++ to DNA, 2) Primer now has more target sites to bind to, but the DNA polymerase has all that PCR product in the way of itself and the template 2a) given that DNA polymerase finds the PCR-template everytime---the primer concentration (eg 20nM) will probably be used up in the first few cycles of PCR amplification (probably 4-5 cycles)--now definitely your Mg++ conc will be overwhelmed. Same thing for the buffer capacity, and possible inhibition of Taq from just too much DNA in reaction.

[danfive]

Originally Posted by jiajia1987

I am sorry to add to this, but I do not get why the concentration of the PCR product in the reaction mixture would be a problem. If the primer used in the first PCR anneals to the template and makes a PCR product, the PCR product should be similar to the template, with the ends similar to the primers used in the first PCR, right? Since that is the case, why would the second PCR be a problem? I am confused.

Of course every reaction can be different. Say you start with very little template DNA--and thus your PCR gives you very little PCR product.

Here you can even add more taq to same reaction and rerun the PCR--so all your primers, say 20nM--are used and give you 20nM PCR product.

[jiajia1987]

Dear everyone, thanks for the explanation. They definitely make things clearer for me. I am so glad there is molecular station!

[evakaranjah]

i have used a pcr product and it gave a similar result as before however, my other samples gave primer dimers and this has been a problem for quite a while now.

[esilva]

I am having similar problems.
what is the concentration of the first pcr that you use?

[davidflora]

[Only registered users see links. ]You would be amazed at how often a PCR reactions SEEMS to produce the correct band, when in fact it has amplified something spurious. Sometimes when you sequence the band, you will discover that the sequence is completely unexpected and nonsensical. At other times, sequencing with one of your PCR primers will give a completely blank lane, while the other primer will give two simultaneous and superimposed sequences.

[Gustaveimj]

ÿþ[

ÿþ[

ÿþ[

[ajitk76]

nice suggestions as I also got same problem and now I will do the dilutions and try it again.

thanks

Ajit India