any ideas for substitute for chloroform

Andre Hamel hamel at cc.UManitoba.CA
Sat Apr 29 00:14:30 EST 2000


So far, my experiments with trichlorobutanol (TCB) is that it makes a
fine additive to
chloroform (CHCl3). The TCB (mp 77*C) is readily dissolved in chloroform
at rm temp.
I found that 20% (w/v) TCB in CHCl3 imparts enough increase in viscosity
to the CHCl3
so as to make it more convenient to aliquot (even with Pasteur pipet).
Progressively higher
concentrations of TCB make the CHCl3 more viscous, but by 50% w/v, the
TCB seems
to be near saturation in CHCl3 (crystals of TCB appear along the edges
of the vessels
(pipets and pipet tips, microfuge tubes, glassware, bottles, etc). Thus,
I stayed with using
TCB at 20% w/v in CHCl3.

The TCB at 20% w/v in CHCl3 stores well at +4*C and -20*C. When stored
below
+4*C, some tiny specks of crystals appear along the walls of the glass
bottle. One merely
has to shake it a bit in order to get it clear again (to redissolve
these tiny specks of TCB).
.
I've tested CHCl3 with 20% TCB for about 100 small scale preps of DNA
and RNA (final
yield 5 to 50 ug) used for PCR and RT-PCR of DNA & RNA viruses &
bacteria. There
were no noticeable difference in yield of nucl acid for about 100 preps
compared with preps
using CHCl3 with 2% n-butanol. Also, all gave same intensity of PCR
product for their
respective counterparts.

All preps involved 4 M GiTC/2% sarkosyl/0.1 M 2-ME/0.2 M NaAc and ext'n
with equal
volume of 50:50 Tris sat'd phenol/CHCl3-TCB, followed by ext'n with
CHCl3-TCB (to
remove phenol) followed by ppt'n in EtOH. I performed the ext'ns on ice.
All stages behaved
the same as CHCl3 counterparts (interface were the same, phases looked
the same). The
only noticeable difference was that on ice, the lower (organic) phase
had slightly more
viscosity using CHCl3-TCB compared to CHCl3-BuOH. IMOH, this is good
because it
makes the organic phase slightly more stable when pipetting off the
upper aqueous phase.

I tested on whole blood, serum, nasal swabs, feces (swine), tissues
(lung, lymph nodes, tonsil,
intestine, spleen), bacterial colonies, cell pellets of liquid bacterial
cultures, cell cultured viruses.
I also tested on whole semen, its cell pellet and its plasma.

Since we do not have a radioisotope license (never had a need for it
since we use PCR for
everything and confirm our PCR products by RFLP and by sending away to
be sequenced).
I am confident that CHCl3-TCB works well. The interface is the same as
its CHCl3 counterpart.

Apparently bromochlropropane is used by some as a substitute for
chloroform (some of
Chomczynski's kits?).  However, I'm extremely wary of using any
brominated compounds for my
nucleic acids because they are extremely sensitive to light and can mroe
readily form free radicals
(HBr), which wreak havoc on nucleic acids and proteins ... such preps
become discolored over
time, despite storage in dark (-20*C freezer). Remembering basic
chemistry, fluorine atoms are
smaller than chlorine, which is smaller than bromine which is smaller
than iodine. Imagine working
with iodinated aliphatics instead of chlorinated? ... one would have to
work in total darkness
otherwise ANY light would create free radicals ... chain reaction ...
destroyed nucleic acids as result.

Has anyone else bothered to give a try at using CHCl3-TCB?

If so, please post your experience to this newsgroup.

cheers,

Andre Hamel


---




More information about the Methods mailing list