Hi all!
I used Turbo DNase (Ambion) to treat my RNA samples for gDNA contamination followed with heat deactivation (as in the protocol from manufacturer). The gDNA is removed but I encounter another problem.. After cDNA synthesis the PCR doesn't work! I checked up all possibilities (RNA degradation, cDNA synthesis fail, PCR products etc), everything leads to Turbo DNase buffer..
To test it, I used DNA template that works and prepare 3 samples for PCR:
1) mix of 0,5ul DNA with 0,5ul of the cDNA samples that does not give results,
2) I added aliquot of DNase buffer to my DNA sample;
3) positive control (just the DNA without any additives).
After standard PCR, only positive control gave good band; the mix between DNA and cDNA showed very faint band and last sample failed completely! Did anyone have similar problems?
I tried to purify my cDNA on a column, precipitate, add betaine, caseine, BSA, adjust Mg2+ concentration, dilute, add 1% Triton-X.. all for nothing.
Only 10x dilution and addition of 1% Triton gave me very faint band.. and in 2 cases (out of 40) precipitation restored template activity.
After column purification or precipitation my samples are clean (perfect spectra on NanoDrop) and of ca. 30 ng/ul concentration. Gels of RNA and cDNA look good too.
I am desperate.
Please help!
-tilia25-
Did you try phenol/chlorophorm extraction instead of column purification? Is your PCR enzyme somehow super sensitive to inhibition?
Anyway we use TURBO DNase-Free kit for a routine treatment of our RNA and we never had problems with inhibition.
-Trof-
I tried phenol/chlorophorm extraction and it didn't change anything..
I am not sure about the polymerase.. We've always used Taq polymerase from Invitrogen and never had problems before. Which one do you use?
Thanks a lot!
-tilia25-
If the DNase buffer is the sole problem, do your DNA digestion in your RT buffer, turboDnase works well in broad ion strenth and pH scale. Good luck.
-WSN-
