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After reading the material requirements in the top, it appears...
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Marco Binder Oct 24, 2013
After reading the material requirements in the top, it appears to me that one step simply has been forgotten in the protocol, which would be the phosphorylation of the CIPped RNA by polynucleotide kinase and gamma-P32-ATP. Then everything would make sense again!
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Bio-protocol Editorial Team Answered Oct 24, 2013
bio-protocol.org
Dear Dr. Putzer and Macro,
The missing step has been added as shown at step 1c. We deeply apologize for our mistake and any confusion that have caused to you.
Sincerely,
Bio-protocol editing team
The missing step has been added as shown at step 1c. We deeply apologize for our mistake and any confusion that have caused to you.
Sincerely,
Bio-protocol editing team
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Harald Putzer Author Answered Oct 24, 2013
CNRS FRE3630, Institut de Biologie Physico-Chimique, France
Dear Marco,
you are correct, the labeling step is not in protocol even though it was in the version that I had submitted. Sorry for that. The missing part is:
3) Labelling reaction (20 µl)
Dephosphorylated RNA 1 µg
T4 Polynucleotide Kinase buffer 10X 2 µl
RNase inhibitor (40 U/µl) 0.25 µl
?32P ATP (10 µCi/µl) 5 µl?
T4 Polynucleotide Kinase (10U/µl) 1.25 µl
H2O to 20 µl final volume
a. incubate 2 hours at 37°C
b. Precipitation as in step (2) and take up the pellet in desired volume
I will ask to correct this on the web site.
Harald
you are correct, the labeling step is not in protocol even though it was in the version that I had submitted. Sorry for that. The missing part is:
3) Labelling reaction (20 µl)
Dephosphorylated RNA 1 µg
T4 Polynucleotide Kinase buffer 10X 2 µl
RNase inhibitor (40 U/µl) 0.25 µl
?32P ATP (10 µCi/µl) 5 µl?
T4 Polynucleotide Kinase (10U/µl) 1.25 µl
H2O to 20 µl final volume
a. incubate 2 hours at 37°C
b. Precipitation as in step (2) and take up the pellet in desired volume
I will ask to correct this on the web site.
Harald
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