Wear gloves throughout and work in radiation area. Monitor area before and after use. Mix the following in an eppendorf tube: 1. 0.5 microgram oligonucleotide dissolved in H2O. 2. 3 microliters 10x kinase buffer. 3. 2 microliters 32 P ATP from ICN (>5000 ci/mmole). 4. H2O so that the final volume is 30 microliters. Add 25 units T4 polynucleotide kinase and incubate 60 min at 37 ℃. Purify labeled Oligonucleotide away from unincorporated ATP Currently, we use mini Quick Spin Oligo Columns (#1 814 397) from Roche to purify the labeled oligonucleotide. Prepare the column according to the manufactuer's instructions by centrifugation of the resuspended matrix for 1 min @ 1000 x g. Insert column into a new eppendorf tube and add oligo labeling reaction, adding slowly to center of column. Centrifuge 1000 x g for 4 min. Recover purified labeled oligo. For most applications, add 70 microliters TE to the 30 microliters recovered for a total of 100 microliters. Quantitate radioactive incorporation by counting 1 microliter of a 1/10 diluted sample. Expect between 20 -100 million cpm total. 10x Kinase Buffer 0.5 M Tris pH 7.6 0.1 M MgCl2 50 mM DTT |
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