Thawing on ice necessary? - (Mar/08/2006 )
Hi there. In so many protocols it says to thaw proteins, DNA, PCR reagents on ice. Unfortunately, they can sometimes take over an hour to thaw. It seems that all the ice does is slow the solid-liquid transition, but is this really necessary? Or is it just to protect someone from forgetting their reagents and letting them get to RT for +20 min. What do you think?
About the only thing I bother to thaw on ice is competent cells...
I agree with homebrew.
I either thaw them in my hands, so i don't forget them at RT, either I let them on ice if I think about it in advance and there is no danger I forget them.
(but I thaw competent cells always on ice)
one thing I do sometimes, is to thaw things that take forever by laying them on top of the ice...they stay a bit cooler than RT but thaw much faster than if they are imbedded in the ice
another thing, when thawing primers, RNA, dNTPs...most NA stuff...I put them in a rack in the fridge. they will thaw pretty quickly but you don't have to worry about forgetting them in the heat
protein preps, nuclear extractions...hmmm...I worry about stability and so thaw them always on ice. comp cells too
I also usually thaw PCR and RT components and NA's on top of the ice, too.
But, I have also thawed PCR and RT components in a sterile, RNase-free, room temperature water bath. That works very quickly and is great if you have a component that you forgot to take out of the freezer with the rest of them. I don't know if anyone else does that, but I heard it was ok to do this as long as you don't forget them in it and put them on ice once they are thawed.
In fact, the person who suggested the water bath said that people who thaw things in ice tend to get demotivated and spend hours drinking coffee waiting for things to thaw. Uh..that was me!!
dNTP and polymerase buffer were the only things i wasnt thawing on ice.....well i guess those are the only ones that would take time to thaw ...never had any problem though....
never thaw dNTP and PCR buffers on ice.
they are aliquoted, then I never have more than 50-100 uL to thaw.
I let them at RT while I prepare every thing.
I am thawing everything in a thermomixer set at 55ºC. a few seconds for the dNTPs, up to 5 minutes for the rest. I never have any pb. According to one of our sales rep, the most temperature sensitive stuff in PCR are the dNTPs, and I am carefull with those. He says he was storing all the rest at RT, including Taq. I wouldn't go that far though. But indeed buffers and MgCl2 are not temperature sensitive (in fact MgCl2 shoould be vortexed for a full minute if frozen because of micro cristals according to the same guy).