Hey there,
I isolate DNA/RNA by using the All Prep DNA/RNA Qiagen Kit (96 well plates). Usually the isolation works fine although I must admit I do not have tremendous amounts of DNA/RNA at the end. However, for the last two isolations (using 16 samples (PBMCs) each), I made a small change in the protocol (still establishing my methods in the lab...): After lysing the cells with RLT lysis buffer, I centrifuge the cells shortly (just a few sec at max. 800xg) and put the lysed cells directly on dry ice overnight. The next day I warm up the lysate (for a few minutes in the 37°C waterbath), centrifuge again for a few sec (just so all liquid is at the bottom of the tube) and follow the protocol as written in the handbook. Since I changed the protocol, I have higher amounts of DNA (measuring with DeNovix without any fluorescence dye or so...) and NO RNA left! Can anyone explain why? The handbook says its ok to freeze the lysate at -70°C for several months. Might it be that the DNA and RNA does not separate properly after freezing therefore I have higher amounts of DNA but no RNA at the end? Do you have any suggestion how to handle the freezing? I need to freeze the lysates since I will only have a few patient samples at the time and need to collect more for isolation...using columns (meaning the AllPrep Kit with 50 single columns) is not really an option because its quite time consuming to process more then 300 samples...
Thank you for your help!
Nicole
The freezing should not be the problem. I usually lyse cells in RLT and keep them at -80 for up to months. I never had problem with RNA isolation after that...
Thank you for your answer! Do you also thaw in a 37°C water bath for some minutes?
No, I thaw the samples at 4 degrees, and just transfer them directly on the column even if they are cold. I never had any problem with that.
Ok thank you! I was talking to a colleague yesterday and thawing in the fridge was also her suggestion...I did not have a lot of RNA at the end of my isolations anyway (meaning before I added that additional freezing step in the protocol), so maybe 37°C for some minutes is enough to degradate my little amount of RNA,...
Hey Paul Chammas,
I have another question regarding your procedure, if I may?
Do you use cryopreserved cells for DNA isolation or fresh ones? What kind of cells do you use?
Thank you for helping!
Nicole
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