Hello,
I have a trouble with a PCR reaction. The thing is, a PCR reaction from cDNA goes well, the first picture. But after purifying and using this DNA for the same PCR to multiple it I get the smears (second picture).
I have tried it three times from the beginning, but the result is the same. The first reaction goes well, the second one not. There was a hypothesis that the DNA was digested during the purification process, but repeated electrophoresis disproved it. I have also tried it with another polymerase and got smear. Another hypothesis - polymerase digests DNA fragments (Encyclo polymerase that I use has an endonuclease activity), so I tried it with Taq-polymerase, but it didn't help. The last one - G-quadruplexes in the DNA structure, but in presence of DMSO in buffer there also were smears.
I have also tried to decrease the number of cycles, the amount of DNA, but unsuccessfully.
So I'm asking for your help and any ideas.
you are over amplifying. 10 cycles of pcr is 1000 times more product. you are amplifying to the point where the prodyct anneals to itself better than the primers and are getting longer pcr products. Try using a dilution of 1:1000 and 1ul of this dilution. set up identical pcr tubes and remove tubes from the cycler at 14,16.18,,,,,,to 24 cycles and 2 of these should give strong clean amplifications and a couple will probably look like these smears but you will know how much to use. Diluting also dilutes incorrectly annealing false amplimers and will give a cleaner product than using undiluted first round product
Have you also considered doing "nested PCR" ? (That's what you may have done, but it's not clear to me.)
"nested PCR" consists of carrying out a first PCR cycle with a pair of external primer; then carry out a 2nd cycle of PCR with a pair of internal primers. You will need 2 forward primers and 2 Reverse primers. We can vary the number of PCR cycles, and test different dilution levels with regard to the substrate of the 2nd PCR.
Dear dr. Paul Rutland,
Thank you for your answer!
We tested your assumption and got something strange. The first two lanes consist of PCR product with 14 cycles, than in 4-5th - 18 and 7-8th - 22 cycles respectively. At cycle 22 a smear appears, but at cycle 18 we did not observe amplification of the target product.
Dear dr. David Lepetit,
We didn't use nested PCR, but we will consider it, thank you for your advise!
Dear Dmitry Fedorov ,
in one sense this is the expected result of weak amplification at few cycles then ok then smearing but these results are too small to be pcr product so they must be primer dimer that is amplifying and using up the primer so tere is no long product being generated . David Lepetit suggestion of either fully nested or hemi nesting the second amplification is an excellent idea which will work or try a re amplification with the same primers as you have done but using agarose gel purified pcr first round product as a means of getting rid of the primer dimer generated in the first round pcr