jpcd0 at mole.bio.cam.ac.uk
Tue Nov 8 09:39:18 EST 1994
Hi, I've just started extracting RNA from murine ES cells to be used for
RT-PCR using the Chomczynski et al 86 protocol. I followed their
suggestion of 2mls solution D per 10cm tissue culture dish and froze my
lysates down at -70C.
Firstly, I pinched a post-docs solution D and he asked me to make up some
more as I used the lot. According to the protocol you need 2M NaAcetate at
pH 4.0. I have tried to make this up but even when I dissolve 27.2g of
NaAc.3H2O MW 136.08 in 36 mls of DEPC H2O (the minimum I can use) I am
left with 45mls of 'room' to pH the solution with glacial acetic acid. The
initial soln is pH 9ish and after adding 45mls glacial acetic acid it gets
down to 4.4.
So how do I get it to pH4.0? Do I really need it that low? I have seen a
modified protocol on the Net using the bog-standard pH5.2. Is that all
right to ensure the DNA stays in the organic phase?
If I really do need pH 4.0, am I better off pH-ing the NaAc with a few
drops of HCl at the cost of Cl- contamination or with 50+ mls of glacial
acetic acid which may keep the Na ions at 2M but surely messes up the
OK- hopefully there is a simple answer to that.
Next, are there any improvements on this protocol around?
I am planning to use 900ul of lysate, 90 NaAc (2M ph4.0!), 900 Phenol
(ph4.2 DEPC H20 equilibrated) and 100ul Chloroform:Iso-amyl alcohol (49:1)
which should just squeeze into a 2.2ml eppendorf so that I can spin my
samples at the reqd 10000g in microcentrifuges.
Is it OK to scale down their numbers? Do I need to do all the steps they
suggest (ie isoprop precipitate, resusp in D, more isoprop, spin, 75% Etoh
wash, resusp in 50ul 0.5% SDS at 65C)?
Is it important to spin at 4C rather than room temp?
Are my lysates OK at -70?
Sorry about so many questions in one post, hope someone can help,
John Dixon Lab 44 (223) 334131
Wellcome/CRC Institute Fax 44 (223) 334134
United Kingdom e-m: jpcd0 at mole.bio.cam.ac.uk
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