Dnase digestion after in vitro transcription - (Dec/11/2007 )
After the synthesis of RNA probe by in vitro transcription, the dna template was usually digested by Rnase free Dnase I in Ribonuclease-protection assay. I wonder why the dna template should be digested? If not, what would happen? I mean, how will the DNA interfere with the subsequent reaction? Thank you so much!
-wangray-
QUOTE (wangray @ Dec 11 2007, 01:07 AM)
After the synthesis of RNA probe by in vitro transcription, the dna template was usually digested by Rnase free Dnase I in Ribonuclease-protection assay. I wonder why the dna template should be digested? If not, what would happen? I mean, how will the DNA interfere with the subsequent reaction? Thank you so much!
The DNA will hybridize to your RNA probe and protect it from RNase digestion. Subsequently your probe might no longer be in molar excess over your RNA and will thus give you misleading results.
-smu2-
QUOTE (smu2 @ Dec 11 2007, 10:08 PM)
QUOTE (wangray @ Dec 11 2007, 01:07 AM)
After the synthesis of RNA probe by in vitro transcription, the dna template was usually digested by Rnase free Dnase I in Ribonuclease-protection assay. I wonder why the dna template should be digested? If not, what would happen? I mean, how will the DNA interfere with the subsequent reaction? Thank you so much!
The DNA will hybridize to your RNA probe and protect it from RNase digestion. Subsequently your probe might no longer be in molar excess over your RNA and will thus give you misleading results.
Sounds reasonable. Thank you! But I am still a little puzzled: since the DNA was double strand, how would it hybridize to the RNA probe? -wangray-
QUOTE (wangray @ Dec 11 2007, 07:59 PM)
QUOTE (smu2 @ Dec 11 2007, 10:08 PM)
QUOTE (wangray @ Dec 11 2007, 01:07 AM)
After the synthesis of RNA probe by in vitro transcription, the dna template was usually digested by Rnase free Dnase I in Ribonuclease-protection assay. I wonder why the dna template should be digested? If not, what would happen? I mean, how will the DNA interfere with the subsequent reaction? Thank you so much!
The DNA will hybridize to your RNA probe and protect it from RNase digestion. Subsequently your probe might no longer be in molar excess over your RNA and will thus give you misleading results.
Sounds reasonable. Thank you! But I am still a little puzzled: since the DNA was double strand, how would it hybridize to the RNA probe?When you do an RNase protection assay, you have to heat the probe and target RNA first to get rid of secondary structure. DNA will obviously become single stranded.
-smu2-
Thanks a lot!
-wangray-