UV-Cross

rogier stugerNOstSPAM at cellbiology.uni-frankfurt.de.invalid
Mon Feb 14 10:06:02 EST 2000


Hi Kerstin,
what type of protein do you use? total cell lysate? purified
protein?
the problem may be nonspecific interaction by partially denatured
proteins, 'cos your samples get too warm in the stratalinker.
I keep em cool by doing the X-link in clipped-off lids of eppies,
floating on icewater. samples about 5 cm from the lamps,
irradiation twice at max energy (about 2 x 5 minutes).
I take it that your labeled DNA is doublestranded? check for
proper annealing, cos ssDNA can bind to lots of proteins. If you
wanna be really sure, clone your target DNA in whatever vector
you like (with restriction sites on the edges), then PCR with hot
dNTPs. choose primers so that the total length of PCR product is
about 100 nt, then cut out your 28 nt fragment. This is a very
easy way to make labeled DNA and you're always sure it's really
double stranded.
Good luck,
Rogier Stuger
Dept MicFizz, Free U of A
E rogier at bio.vu.nl


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