PCR reaction ratios

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PCR reaction ratios

Postby lcini » May 10 2017 10:24 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 11:27 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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Re: PCR reaction ratios

Postby 29yrsExperience » Sep 07 2017 6:30 pm

A lot depends on the purpose of your experiment. If you are doing anything remotely quantitative, this requires a do-over. Likewise for endpoint PCR- if you looking for presence/absence of a target in your template, this is not good science, even if it “works.” However, if you are amplifying a known target in your template for the purpose of cloning it, and intend to sequence it once it is cloned, and you know exactly what the sequence should be, and you get a nice band of the expected size, (sorry for all the “ands”)-I’d say go ahead and clone it. Sequencing will be the ultimate confirmation that it is OK.
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