5' end labeling of tRNA

Narasimham Jammi jammi at SELWAY.UMT.EDU
Wed Apr 5 12:14:09 EST 1995


Hi!
I've been trying to 5' end-label tRNA-phe (yeast) from SIGMA. I have been 
able to end label it, but ever so slightly. I treat tRNA with Shrimp 
alkaline phosphatase ( I've done this with different amounts of SAP for 
varying periods of time). I inactivate SAP for 15 min at 65 C as per the 
manufacturer's recommendation. Then I add diluted PNK at 3 units/ul
directly to the tube without phenol extracting off the SAP (I have done 
it the usual way as well - but I lose a lot of product this way!). 
Incubate for 1 hr @37 C and inactivate PNK for 5 min (again as per the 
instructions). Analysis of an autorad shows a very thin band where the 
tRNA is. So here's my question - How do I optimize my labeling 
conditions?. Does it have something to do with the 5' recessed end of 
tRNA? And btw, this expt. works great for upto 100 picomoles of tRNA. But 
when I have greater concns. (like say, 760 pmoles), I have very poor 
yields. Any info about the stoichiometry will be greatly helpful and 
appreciated.
Thank you,
jammi
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