re zebrafish mRNA preps
clark at EUROPA.LIF.ICNET.UK
Tue Nov 15 11:59:50 EST 1994
i also have just started a project which involves the isolation of mRNA.
i have tried shield and 26 somite stages so far and two protocols.
for total RNA I used a modification of AGPC (acid guanidine phenol
chloroform) method of CHomczynski and Sacchi.~2.5mls of embryos yield
700ug of total which gives ~7ug of polyA+.(From 26 somites stage).
polyA+ selection was done using In Vitrogen Fasttrack mRNA kit, this can
be used to isolate mRNA directly from embryo's but 1g/1ml is all you can
use at once, and it was my first time with RNA i isolated total first
checked the quality, and then proceded to polyA+.
i have found that you can reduce the volume of embryo's by ~3x just by
dechorionating using pronase, which i do just before freezing in liquid
nitrogen to minimise any possible effects.
so for my next prep i will try direct to polyA+ from dechorionated embryos.
let me know how you get on and feel free to askm any more questions
p.s.the polyA+ i have isolated so far has been of good quality andi am
now proceeding to make a cDNA library from it.
Genome Analysis lab
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London WC2A 3PX
+44 71 269 3447
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