3'-end labeling of tRNA
SURESH KUMAR K.G.
skg at biochem.iisc.ernet.in
Tue Jul 3 13:12:42 EST 2001
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
happy labelling...I have been through lots of tRNA end labelling too..
Suresh Kumar K.G
Dept of Biochemistry
Jul 2001, January Weiner wrote:
> 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?
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