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Problem amplifying plasmid


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#1 MaggieRoara

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Posted 14 October 2009 - 09:02 PM

I am having a really difficult time with troubleshooting a PCR reaction thats not working. I am not seeing any PCR bands. I have narrowed it down to the problem either being because of the primers or the plasmid template. Is it possible for thereto be something wrong with my template. I checked it on a gel, there's a band at the correct place i.e. the miniprep to get the template was successful.

#2 gangut

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Posted 15 October 2009 - 06:15 AM

what about digesting the plasmid (outside of the amplified region of course) to prevent it from supercoiling? Too little and too much of template can inhibit the reaction as well




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