After synthesising cDNA today I noticed some residue on the seal and on the walls. Most of the residue on the top, on the seal, came down upon centrifugation... However om The walls the tiny specks remained and wouldn't return to the bottom regardless of spinning a second time. I'm worried this will affect my qPCR results. I did try to move them down physically using pipette tip but the specks were so tiny and felt almost stuck to the walls. Has this happened to you and why does this happen?
Hi Carrie, of course, that was salt from your reaction buffer that crystallized on the walls of the tube after the water evaporated during the reaction.
Suggestion: Mix thoroughly and briefly spin your tubes BEFORE you incubate them for the RT reaction.
If you also wish to avoid evaporation on the lid (but that will be only water which you can recover as you already did, although it means your reaction slowly concentrates in time) use a PCR tube for the RT reaction and incubate in a thermocycler using the hot lid.
Good luck.
Hi Carrie,
As Pietro said, that tiny specks are crystallised buffer as water evaporated during the RT reaction. You can avoid this by doing a short spin of your PCR tubes prior to the cDNA synthesis. There shouldn't be any drops in the walls.
The crystallised buffer shouldn't interfere with your qPCR, but if you can, repeat your experiment and compare the results.
Good luck!
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