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PCR amplification of a promoter sequence for gel extraction worked beautifully using Phusion HF enzyme with GC (higher error but less picky) buffer. However, DNA concentration from the gel extraction was too low, so I need to do it again. Now with all the reaction parameters the same it is not working. So far I am using the same Phusion enzyme stock and buffers, the same primers which worked before, the same proportions, and the same thermo-cylcer set up. I have extracted fresh genomic DNA to amplify from thinking maybe the DNA had degraded with no luck. I make a mastermix fresh before each reaction by combining the Phusion enzyme, nuclease free water, dNTPs from a 10mM stock, and the GC buffer mix. Any ideas would be appreciated.

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  • $\begingroup$ Did you scale up the reaction to get more yield? $\endgroup$ – Chris Dec 2 '15 at 20:19
  • $\begingroup$ Yes, I have gone up to 50ul reactions and load 20ul samples onto the gel each time for the gel extraction. $\endgroup$ – drtran Dec 2 '15 at 20:20
  • $\begingroup$ Don't do that. You change the characteristics of the reaction - bigger volumes heat up and cool down slower. Instead of doubling the reaction volume, rather do two reactions. This usually works better. $\endgroup$ – Chris Dec 2 '15 at 20:58
  • $\begingroup$ When I scaled up I did use thermo fishers tool to recalculate the proportions for the mastermix. I will keep this in mind when trouble shooting in the future though. Thanks $\endgroup$ – drtran Dec 2 '15 at 23:11
  • $\begingroup$ Have a look at this answer here at bio.se. That's what I have in mind. $\endgroup$ – Chris Dec 3 '15 at 7:16

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