Good poly-A+ isolation kit, anyone???
paraic at icr.ac.uk
Tue Aug 8 14:12:52 EST 2000
I've used both Fasttrack from Invitrogen and Quickprep from Pharmacia
with variable success (FastCRAP, and pretty OK respectively) but now I
use Oligotex from Qiagen and it works really well in my hands. Very fast
protocol, and with great yield and A260/280 and no ribosomal bands. A
bit more expensive, but worth it for the end product.
"Mr B.J.H. Jansen" wrote:
> Dear BioNetters,
> 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
> The Netherlands
> Tel: +31 (0)24 3617239
> Fax: +31 (0)24 3541184
> Email: basjhj at mailbox.kun.nl
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