3'-end labeling of tRNA

SURESH KUMAR K.G. skg at biochem.iisc.ernet.in
Tue Jul 3 13:12:42 EST 2001


Hi J.,

Not an answet to ur qn...but [32-P]pCp is available from NEN life
sciences, or may be u can check with the company from which u get
gamma-ATP. If it is just for labelling tRNA, I think its better u can buy
a vial of [32-P]pCp..save ur time...well..I see from my catalogue
here that NEN has an office in ur country too...check it out...no
affiliations whatsoever...:)
happy labelling...I have been through lots of tRNA end labelling too..
good luck.

regards
Suresh Kumar K.G
Dept of Biochemistry
IISc, Bangalore-12
India







On 3
Jul 2001, January Weiner wrote:

> Hello,
> 
> 	I am labeling  the 3'-end of tRNA using [32-P]pCp. Or, rather, I am
> 	planning to do it. 
> 
> 	Some questions:
> 
> 	1) do you have a nice protocol for making radiolabelled pCp from
> 	3' cytidine phosphate? I have a following one, don't know whether
> 	it is OK:
> 
> 	speed-vac dry 100uCi gamma-ATP
> 	+2 ul pCp-mix
> 		pCp-mix: 3ul 1M Tris ph 8.0
> 		2.5 ul 0.1 M MgCl2
> 		1 ul 0.2M DTT
> 		2 ul 20mM spermidin
> 	+1 ul 1mM Cp (cytidine 3'-phosphate)
> 	5U PNK
> 	---
> 	30' @ 37 deg, 2'@ 100 deg
> 	speed-vac dry
> 
> 	Would you use just standard PNK buffer? (end conc. 50mM Tris 7.5, 10
> 	mM MgCl2, 5mM DTT, 0.1mM spermidine)
> 
> 	2) where do you buy 3' cytidine phosphate? 
> 
> 	Regards,
> 	j.
> 
> 

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