Is 5 min extension time really necessary for 400bp fragments? We’ve been trying to do the whole protocol in one day and it’s been stretching to 13 hours. If we can save time at any step, that would help!
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Unfortunately yes the annealing/extension times are typically long for multiplex PCR so it takes about 4hrs. There are ways to save time though such as removing the PCR cleanup/normalisation that we are working on validating for the next release though.
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I can see how the rate of annealing in multiplex PCR would be slower because of the relatively low primer concentration. But what’s the rationale for longer extension times? Once the primers are annealed to target, I would think that longer extension times would be unnecessary (unless the primers continue to anneal during the extension step). Any related thoughts on this topic?
It’s the latter - the primers continue to anneal during the extension step because it is a 2-step PCR (98°C 15sec, 65°C 5 mins). The actual extension time would be probably 10 or 15 seconds.
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I haven’t worked on a ton of samples and haven’t done any direct comparisons but I’ve had nice success with a 2 minute extension time instead of 5 minutes for PCR