rjordan at u.washington.edu
Fri Sep 4 15:17:45 EST 1998
>Is anybody have a current working protocol to analysis the length of the
>telomeres by a Southern technique.
>Many thanks for your help
Here's the protocol I use. It seems to work pretty well. If you're curious I
can email you a scan of our of our films.
Digest DNA with HinfI and RsaI. I use Gibco enzymes and do a double digest with
the RsaI buffer. Run 1.5ug of each digest on a 0.7% gel. You'll
probably want to determine the time yourself. I run the gel until the
bromphenol blue from the loading buffer is at the end and running off the gel.
Dry the gel. I use a BioRad air dryer, but the person we got the protocol from
originally apparently uses a vacuum dryer. Since it's an agarose gel, I
haven't had too much trouble with them cracking.
Denature the dried gel 2x30min in 1.5M NaCl, 0.5M NaOH, then
neutralize the gel 2x30min in 1.5M NaCl, 0.5M Tris, pH 8.0. The gel will curl
up and eventually come off the cellophane during this step. As soon as it comes
off the cellophane it should uncurl. After it comes off the celophane you can
handle it with a couple pairs of forceps.
Prehyb gel in hybridization buffer at 37C (see end) for 15-30 min. Often I'll
prehyb while I'm preparing the oligo.
Replace hybridization buffer, add labeled oligo and hyb overnight (12-18hr) at
Wash 3x10min in 0.24X SSC.
Expose 24hr at -70C with BioMaxMS film w/intensifying screen.
Apparently using the dried gel gives a better signal than blotting. You can
use radioactive markers to determine the telomere length or stain the gel with
etbr and take a picture with a ruler.
The probe--I use a (CCCTAA)3 oligo that we had made. I label 100 pmoles with 5
ul of gamma-32P-ATP (not dATP) (3000 Ci/mmol) and 30U of kinase. I know that
seems like a huge amount, but that's what the person who sent us the protocol
said to use. You may be able to use less. I've used both Promega T4 kinase
and BM E coli kinase without any problems. After labeling I clean up the probe
by spinning it through a Pharmacia G25 spin column. I typically get 20-30% of
my activity out (according to our Bioscan monitor).
5X SSC, 5x Denhardt's solution, 1x pwash (2.33 g sodium pyrophosphate, 26.8 g
sodium phosphate dibasic heptahydrate in 100 ml water)
The pwash is difficult to make. Try heating the water if you need to.
University of Washington Medical Center
Radiation Biology Laboratory
Seattle, WA 98105
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