Radioactive probes by PCR
gold at astro.ocis.temple.edu
Mon Dec 26 14:37:48 EST 1994
Recently I prepared a radiolabeled probe from a 400 bp PCR fragment
1) Purifying the fragment from an agarose gel (I use gene-clean,
but whatever method you normally use will probably work).
2) Assess the amount of DNA fragment by loading 1/10th on a minigel.
3) Random prime the appropriate amount of the fragment (I think I
used about 75 nanograms) using the Amersham Multiprime labeling
kit #RPN1601C and alpha 32P-dCTP from NEN/DuPont.
4) I purified the labeled fragment from the unincorporated nucleotide
by annealing it to gene-clean (Bio 101). This works well, but
you must use three washes. I'm sure a minicolumn will do...
5) I ended up with between 75 and 100 million DPMs of label.
It worked well on a Northern.
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