2 questions about kinase and phosphotase

Zhong Silin via methods%40net.bio.net (by stxsz1 from nottingham.ac.uk)
Thu Nov 8 12:51:18 EST 2007


Hi
 
I am going to label the 5' end of a synthetic RNA (from T7 in vitro trans). The first nucleoside (normally a G?) should has 5'-triphosphate, although I remeber some ppl told me a lot of the 5'-ppp were truncated during the in vitro trascription. 
 
question 1
 
Since the polynucleotide kinase PNK shouldn't be able to phosphorylate the intact RNA with 5'-triphosphate in a forward reaction, can the exchange reaction work? I know the exchange reaction can transfer a 5'-phosphate group to ADP. It should not work on the 5'-triphosphate.
 
question 2
If neither forward nor exchange reaction can label the RNA, can I dephosphorylate it using a phosphotase (CIAP) and then label it with PNK? The same question is: can CIAP remove the triphosphate.
 
I know it is silly to label a synthetic RNA. If I use hot NTP in the in vitro transcription, it won't need to be labeled. But it is a long story.
 
Thanks in advance
 
 
     

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