I had stored index PCR products of 6ul in -20°C in PCR plates. The plates were covered, not sealed though. Now, after two months, I have to take out 2ul from it to repeat sequencing of some samples. The products now look like tiny crystals stuck to the bottom. Out of the 96 wells, two have stayed as liquid but all the others as these crystals. Does anyone why this could have happened and also, is this bad news? Are my products gone forever or can I dissolve them in a buffer and send it for sequencing? If yes, what should the buffer be?
A picture of how my products look like now in tubes is attached
Your samples are fine. Probably in storage the small temperature difference between the top and bottom of the wells means that the water has sublimed and frozen on the sides and inside top /seal on the tube. Most likely if you thaw the plate and spin it down the 6ul of water will again appear on the bottom of the tube. Warming the tube will then redissolve the dna which is very stable especially when freeze dried which is what has happened to your samples. If the water vapour has escaped then just add 6ul of water and redissolve the samples by warming to 50c for 30mins approximately. What you are seeing happens in all freezers but is especially common in self defrosting domestic type freezers where the frosting on the heat exchanges is removed by circulating warmed refrigerant round the heat exchangers to melt the frost and samples sitting on the exchangers get heated temporarily
I would recommend resuspending and running them on a Bioanalyzer and using a Kapa library quantification kit (https://rochesequencingstore.com/wp-content/uploads/2017/10/KAPA-Lib-Quant-ILMN_9.17-IfU_1.pdf). This will let you know after resuspending if you still have intact library and at what concentration your libraries are at.
It's just evaporation. Thaw them, spin them down, remove the cover, and then adjust the volume back to 6 microliters. You should be just fine.
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