I want to know, if I keep PCR amplified products at -20 degree centigrade for overnight, and then I further process it before bacterial cloning. Will it affect the cloning of the bacteria? What should be done to improve its efficiency?
Regarding TA-Cloning it´s best to carry out PCR and cloning on the same day (directly after PCR make a clean up, then clone). Unless you make an extra a-tailing step.
Try cloning with freshly generated amplicons. Unless necessary avoid storage as indicated for TA cloning where u req the A Overhang
Fresh is best and maybe if its only overnight 4°C might be even better as you avoid freeze-thawing.
I prefer fresh but -20 C is ok and if it is overnight don't worry at 4C.
According to "Laura B Geyer (· 18.50 · 42.39 ·), Smithsonian Tropical Research Institute: PCR products are incredibly stable - even more so than genomic DNA. I've had products that were stored at room temp in a bench drawer for several years which I was able to both direct sequence and TA-clone with no problems. The only thing to maybe avoid is multiple freeze-thaw cycles (which can cause shearing), so if you're going to be using it a lot in a relatively short time period it's probably better to go with 4oC refrigeration instead of freezing. Although even there it's more a problem with genomic DNA than PCR products."
-20 Degree Celcious is OK for 1 to 2 days. But 4 degree is ok for week. I checked it in my experiment and found Ok for amplified product.
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