PCR on PCR product

[aftabac]

Hi

According to my personal experience when you do PCR on PCR product by using the same primer pair, you use in first PCR you can't get any product in second PCR. do any other person have the same experience? if yes then what are the possible solution for this?

regards
aftab

[Aga]

Yes, it must be something with 5' and 3' ends - maybe some degradation? I have the same experience. The only thing to overcome this is to do nested PCR.

[danfive]

I've had the opposite experience.

Considering that you have strong (well-designed) primers, they will stick to the PCR template product---the unit-length amplicon--and allow for DNA elongation.

I've used this PCR product or amplicon as template for PCR synthesis of ISH DNA probes (same PCR but with DIG-nucleotides), to bring up a suspect weak diagnostic PCR signal; and for template for nested and semi-nested PCR.

In my experience PCR product serves as fine template and I've used a dilute PCR reaction as positive control very effectively.

The only issue is not overloading a PCR reaction with too much of the DNA product---I recall making dilution of 1/50 (30-35 cycle PCR) and even 1/1,000,000 for a 40-45 cycle PCR.

[Aga]

Thanks danfive, I may try to use your experience in my work.

[ted9999]

You may want to check that your primer is not annealing to a different position during the second PCR. In other words, make sure that the oligonucleotide sequence is complimentary to only one position and not very similar to another.

If the primer anneals in the wrong position it maybe due to the GC content (primer maybe different than any other location, but similar enough to H-bond in the wrong position) and primer lengths.

[aftabac]

Hi
i did experiment with diluted PCR product. i made 100, 200 and 2000 dilution of PCR product and run PCR reaction on PCR product and it worked and i got the results.
thanks for your suggestions.

regards
aftab

[danfive]

Originally Posted by aftabac

Hi
i did experiment with diluted PCR product. i made 100, 200 and 2000 dilution of PCR product and run PCR reaction on PCR product and it worked and i got the results.
thanks for your suggestions.

regards
aftab

That's great!
Now watch out for aerosolized PCR amplicon contaminating your lab.

[ted9999]

Congrats!

[aftabac]

Hi
i discussed this issue with some of my colleagues and they were having the same problem in PCR. so its nice way to sort out of problem. we discussed that theoretically we should get the product but practically we are not getting, and it was due to more cocentration of PCR product in reaction mixture.
anyhow thanks for valid and nice suggestion


regards
aftab

[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.