PCR reaction ratios

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PCR reaction ratios

Postby lcini » May 10 2017 5:24 am

I am wondering what will happen if my reaction ratios are off. I accidentialy put water + template into my template, and nothing into my negative control (besides master mix (obviously)) and I am wondering if this whole reaction is a fail or if the negative control with out the added water for volume will still count as a true contamination control, or is the reaction automatically not going to work because the volume is off (10ul of water missing out of a 25ul total reaction volume).

Likewise for the template + water - I know this throws the ratio of polymerase + primer to DNA off, and now this reation was 35ul instead of 25ul.

I will probably just run the gel and see what happened, but I thought I'd throw it up here in case anyone does know the answer.

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Re: PCR reaction ratios

Postby mdfenko » May 10 2017 6:27 am

the reaction should run, although not as efficiently as normal since the ionic strength (and mg concentration) are lower than ideal for the mix.

the negative control will only tell you if the mix is contaminated, not the water. and the ionic strength will be higher than ideal.

running the gel, as you intend, will give you answers.
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