I just had one question regarding the possible effect of putting to much Taq polymerase in my PCR tube?
Instead of 5µl I put 50µl (10x more).
Do you think it will have a bad effect on the reaction??
Thank for your help.
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$\begingroup$ I'm voting to close this question as off-topic because it is a question of no general interest to the Biology community, which is what this site is trying to accumulate. $\endgroup$– DavidCommented Oct 10, 2019 at 21:03
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$\begingroup$ @David, I think it is valid question for newcomers. $\endgroup$– DexterCommented Oct 14, 2019 at 12:24
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1 Answer
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Costly error. And yes, it will affect your reaction, since the concentration of Magnesium ions, which are needed as a co-factor by the enzyme, is too low. If you need so much PCR product, you can now adjust the rest of the reagents or you could start over and put this on your personal list of errors. Always check the amount of volume, if it is suspiciously high, check again.
If you decide to proceed and work with a 10x larger volume, make sure that the single PCR reaction is not bigger than 50µl, because the bigger volume also affects how fast the reaction heats up and cools down. If your reaction is too slow to reach 72°C for example, the elongation time might not be long enough to get the full product. If denaturating is too short, your efficiency will drop.
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$\begingroup$ Thank you very much for all these explanations. $\endgroup$– beanCommented Oct 14, 2019 at 6:27