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DNA sequencing question help


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#1 claritylight

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Posted 07 September 2010 - 03:25 PM

I use pGEM as a positive control in my sequencing reaction and it helps me to basically see my sequencing reagents are optimal and working. But since it is a plasmid and the DNA I'm actually sequencing is usually human, I have to make up another mastermix for pGEM separately. So I have two mastermixes, one big one for all my human sequencing reactions, and a small one for pGEM plasmid sequencing controls. They are different volumes of reagents for each mastermix (see below). The disadvantage to this is that if my reactions failed, but my pGEM passed, I have to start looking at all the reasons why the failures. But if pGEM was also made up as part of the same mastermix as my human DNA, I could at least rule out I made up my mastermix correctly for the human DNA sequencing reactions.

My question is how to make up pGEM control as if it was for human sequencing mastermix, not for plasmid sequencing mastermix? So that I can just make up a normal human sequencing mastermix, and add pGEM plasmid DNA into it, instead of having to make up a separate mastermix for plasmid DNA?

Human sequencing mastermix
reagent volume (1x)
5x buffer 1.83
DMSO 0.25
primer 0.06
seq dye 0.18
DNA 2.7

Plasmid sequencing mastermix
reagent volume (1x)
ddH2O 1.5
5x buffer 3.66
DMSO 0.5
primer 2
seq dye 0.36
DNA 2

#2 mdfenko

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Posted 08 September 2010 - 11:59 AM

you can make one mastermix with everything but dna, primer and water. then add partial mastermix, primer, dna and water to final volume for each reaction.
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