The polymerase system I am using is Thermo Phusion Hot-Start II, a pfu-based recombinant Pol with a reversible affibody. The reason that I don't want to purify the reaction mixtures after thermocycling is that the target product is only 70 bp, which is too small to be retained by most spin column systems (the exception being the Qiagen Nucleotide Removal Kit, but in that case, 70 bp is the bottom limit, right at the cut-off). I've been gel extracting the desired band, but for work-flow reasons, I'd like a better idea of how long my un-purified reaction mixtures can be stored at -20C (or -30C, or -80C) before being gel-extracted.
My instinct is that the reversible affibody and low temperatures (combined with loss of enzyme activity due to damage from freezing) will prevent the enzyme's proofreading domain from significantly degrading the target product. The real concern, then, is that the buffer conditions may not be ideal for storage. Thermo won't disclose their buffer composition (trade secrets and all), but I can't imagine that it would be damaging in the short term. Any thoughts are appreciated :)
In principle, for several months. But if you want to prolong the storage more than six months, I advised to store the samples in -80. Hope this help.
The product can be stored for more than 6 months provided you keep your product at -80 0C. But don't freeze thaw the samples. if you have to thaw my advice is to keep aliquots
Dear Dr Brendan Delange,
Thank you for your question. I think you can keep your sample for only short time at -20 or -80C better. but you do not need longer after finish your running in gel.
But you must take care at low temperature may be evaporated or contaminated
with best regards
Khaled
Thanks all! I will only be storing my PCR samples for a short time before gel extraction - a week at the most, so it sounds like I shouldn't have any problem at -30C for that time.
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