Good poly-A+ isolation kit, anyone???
Mr B.J.H. Jansen
bjansen at hgmp.mrc.ac.uk
Mon Aug 7 06:35:37 EST 2000
Currently I'm in the process of constructing some good old c-DNA libraries.
The input m-RNA comes from cultured keratinocytes, and the problem is that
these do not grow to high density (in Keratinocyte Growth Medium up to 5*10^5
cells per standard dish of 20 cm^2), which means that I've to use an awful
lot of lysis buffer because of the large surface area on which these cells
grow. I have to isolate total RNA first, and yields are fairly consistent.
So far I have used kits from Pharmacia and Roche to further purify poly-A+
RNA, with widely varying results. What bugs me is the 'widely varying'part,
and I was wondering whether one of you has ever compared various poly-A+
isolation kits. I have so far isolated poly-A+ from 50 to 100 micrograms of
total RNA, admittedly not very much, but in one case it has already resulted
in the birth of a c-DNA library. Oh yeah, since we try to pick up genes that
have been identified in SAGE libraries, commercially available c-DNA libraries
are not an option. Thank you for your help!
All the best
Bastiaan JH Jansen
Dept of Dermatology 802
University Medical Centre St Radboud
PO Box 9101
6500 HB Nijmegen
Tel: +31 (0)24 3617239
Fax: +31 (0)24 3541184
Email: basjhj at mailbox.kun.nl
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