Will ATP inhibit Reverse Transcription? - (May/25/2007 )
I want to ligate a linker to a short RNA sequence.
For this I will use T4 RNA Ligase1 (ssRNA Ligase from NEB).
Usually this step is followed by a Phenol:Chloroform extraction and ethanol precipitation.
In order to minimize losses of sample I want to avoid that step and move directly to Reverse Transcripion.
If I compare the contents of T4 RNA Ligase1 Buffer and the Reverse Transcriptase buffer, they look almost the same.
1mM ATP is the only difference. ATP is present in the T4 Ligase1 Buffer but not in the RT buffer.
My skills are limited so I dont know if the ATP will interfere in some way with the Reverse Transcription process.
I would be very thankful if somebody could give me a hint or explanation about this issue.
T4 RNA Ligase 1 Buffer:
50 mM Tris-HCL
10 mM MgCl2
10 mM DDT
1 mM ATP
Reverse Trascription contents:
First-Strand Buffer - 250mM Triss-Hcl (pH 8.3), 375mM KCl, 30mM MgCl2
100 mM DDT
10 mM dNTP
PowerScript Reverse Transcriptase
Thanks for reply.
have a close look...
dNTP is a mixture of all four nucleotides, that means there is also dATP in that buffer.
dATP is in that buffer, but not ATP...
(don't know the answer, sorry).