RNA from skin tissue

Andre Hamel hamel at ccu.umanitoba.ca
Fri Apr 16 19:21:53 EST 1993


Try simply adding some skin to at least 10 volumes formamide, vortex well,
then boil for 10 min, vortexing inbetween couple of times, then simply PCR
aliquots (1 to 10 uL in 100 uL rx'ns .. Taq pol WON'T work for this
though, you need to use Pfu, Tth or Vent polymerases) .. remember to
DECREASE the annealing and polymerization steps of the thermocycles by
appropriate # of oC, for example, if 10 uL formamide lysate is used in 100
uL reaction, then the annealing and polymerization steps ought to be
lowered by about 10oC. Assuming PCR product size of about 500 bp, maybe try
95oC for 5 min once, then for 30 to 50 cycles, using 95oC for 30 sec,
annealing temp for 15 sec, then polymer. temp for 15, increasing to 120 sec
by the final cycles, then hold at polymer. temp for 5 to 10 min.

regards

Andre

In article <1993Apr16.182033.1 at vaxa.strath.ac.uk> cdds01 at vaxa.strath.ac.uk writes:
>
>Dear Netters,
>
>My good lady is attempting to PCR cytokine genes from  skin, however
>she is not having much success preparing the tissue. She is trying to
>grind it up in a morter and pestle, in liquid nitrogen, but the tissue
>refuses to break up, it just stays in one piece and stretches (so to
>speak).
>Anyone out there know how she can overcome this problem?
>
>Pete Crilly



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