At 03:43 PM 10/9/96 GMT, Dr. Randal W. Giroux wrote:
>We are adding some linkers to our vector to change the restriction site
>and we have to phosphorylate one of the two oligos (16mer) before we
>ligate them to the vector.
>> BUT once we finish the reaction with EDTA we need to use these oligos in
>the next ligation reaction. The t4 kinase enzyme and EDTA are still around
>and the EDTA will inhibit ligation and the T4 kinase will still be active.
>How can I purify the oligos away from the phosphorylation reaction
>components. Are there kits or quick protocols to do this. I'm stumped
You don't have to get rid of the EDTA and the Kinase may be heat-killed at
70 C for 15 min.(EDTA is not needed to inactivate the kinase by heat) Just
add molar excess of Mg++ to the reaction to counter the effect of EDTA.
Of course you could make use of a desalting/buffer exchange column.
Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu