I have some frozen RNA samples in -80 and I want to do cDNA synthesis fallows qRT-PCR. I am using DNase I from genei
i want use EDTA for deactivation of Dnase I after treatment with RNA
thank you..
We use the DNase I kit from Invitrogen. That one has an EDTA deactivation step, using 1uL of 25mM EDTA for each 10uL sample.We get good results with the cDNA synthesis after we've used this DNase Kit. Depending the quality of your RNA you may need to do this protocol twice though.
The protocol we use:
To each tube:
1ug RNA
1uL DNase buffer
1uL DNase I enzyme
DEPC treated water that make sample add to 10uL
-Incubate the tubes for 15mins at room temperature. Then inactivate the DNase I enzyme using 1uL 25mM EDTA for each of your tubes. Finally, heat the tubes for 10mins at 65 celsius. Samples are then ready to use for cDNA synthesis.
Hope that helps!
see below:
http://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/ampd1bul.pdf
http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/amplification-grade-dnase-i.html
http://arcticzymes.com/technology/double-strand-specific-dnases/
We use the DNase I kit from Invitrogen. That one has an EDTA deactivation step, using 1uL of 25mM EDTA for each 10uL sample.We get good results with the cDNA synthesis after we've used this DNase Kit. Depending the quality of your RNA you may need to do this protocol twice though.
The protocol we use:
To each tube:
1ug RNA
1uL DNase buffer
1uL DNase I enzyme
DEPC treated water that make sample add to 10uL
-Incubate the tubes for 15mins at room temperature. Then inactivate the DNase I enzyme using 1uL 25mM EDTA for each of your tubes. Finally, heat the tubes for 10mins at 65 celsius. Samples are then ready to use for cDNA synthesis.
Hope that helps!
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