BSS027 at uk.AC.BANGOR wrote:
: HELP! We have constructed a mammalian cDNA library and now find that
. . .
: problem and can suggest methods of reliably getting rid of all of
: the DNA from the mRNA prep. We are currently using a guanidinium thiocyanate
: extraction procedure for total RNA followed by purification on an oligo dT colum
: We have tried treating with DNase but the RNA degrades as well!
I have been isolating RNA from isolated nucli by the hot acidified phenol
method, but in the final RNA there is always some DNA. However I have been
digesting the DNA with RQ1DNase (from Promega). I use an invitro
transcription buffer which is known to be RNAse free and add the DNAse and
digest at 37 C for 0.5 to 1.0 hour. I don't seem to have a problem with RNA
degradation as assayed by Northern blot anaylsis. (My hybridizing RNA
shows a sharp band). THe RQ1DNAse is gaurantied to be RNAse free and I
beleive it is routinely used in the digestion of plasmid DNA after an
invitro RNA transcription reaction.