I am trying to do rep-PCR (ERIC/REP/BOX) in E. turcicum and the published article doesn't have the elongation phase just prior to holding. Will it work?
The final extension step will be necessary when your objective is to clone the pcr product into TA cloning vector, since this will be help full in incorporating A residues at 3` end of the pcr product.
Yes, The step is actually not very necessary.The PCR wont be successful if previous stages of amplifications were not completed. So last elongation needs to be only as long as previous steps.Keeping the sample for last elongation may increase the yield a little bit. I never do the final elongation for my PCRs. (5kb fragments, Primestar polymerase, eppendorf thermocycler)
though last elongation phase is not necesary, it is better to go through that step to make your amplication more convenient.
well thanks everyone, it is running as we speak I will see the results in the morning, thanks for your inputs
Yes of course Lebogang, this last step is sort of voodoo anyhow. It is not the last step that decides on success. It's more all the steps before! I never did it in my life and never had problems with analytical PCR experiments nor with preparative PCR with fragments meant for cloning.
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The final extension step will be necessary when your objective is to clone the pcr product into TA cloning vector, since this will be help full in incorporating A residues at 3` end of the pcr product.
As mentioned by others final extension is essential for cloning . It helps in completing the extension of all those reactions that were primed but not completed in the given time for extension in the cycle of denaturation, annealing and extension .
Yes, of course PCR including cloning works without tat final step. Remember: Extension must have worked throughout all amplification cycles, otherwise primer binding would not have been possible. Why should especially the very last cycle behave differently? The only arument for prolonged last cycles could reside in providing more time for adding one additional nucleotide by the terminal transferase activity of the polymerase. In practice, however, this has never made a difference for cloning. I also strongly recommend to add linker extensions instead of the A-addition technique. It works by far better.
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