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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.

drtran
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  • Did you scale up the reaction to get more yield? – Chris Dec 02 '15 at 20:19
  • Yes, I have gone up to 50ul reactions and load 20ul samples onto the gel each time for the gel extraction. – drtran Dec 02 '15 at 20:20
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    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. – Chris Dec 02 '15 at 20:58
  • 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 – drtran Dec 02 '15 at 23:11
  • Have a look at this answer here at bio.se. That's what I have in mind. – Chris Dec 03 '15 at 07:16
  • You can try performing the reaction again using the purified promoter sequence from the first amplification as template. With the low error rate of polymerases like Phusion, the chance of an error occurring for a ~1kb amplification is low enough to not be a significant issue. – March Ho Dec 08 '15 at 09:22

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