CTAB HELP

Rafa Maldonado Rafael at genetics.med.utah.edu
Tue Feb 8 19:43:15 EST 1994


Hi! Here is the protocol used by me. It is a derivative from Protocols
in Molecular Biology (Ausubel FM, Brent R, Kingston RE, Moore DD,
Seidman JG, Smith JA, Struhl K (1987) Current protocols in Molecular
Biology. John Wiley & Sons, New York).
CTAB is very useful to remove polymers like sugars, proteins and other
stuff from DNA. BUT... if you don't adjust very well your NaCl
concentration in every step, you can PRECIPITATE the DNA with the CTAB
if the NaCl concentration is too low. Some people use this method to
precipitate DNA, if you are interetested in it, search in the Current
Protocols in Molecular Biology.
I use this protocol for bacterial DNA extraction (one dumb bacteria
with many poly-sugars and poly-beta-hydroxibutirate, which inhibit
restrictions enzymes) and it works fine.

CTAB/NaCl solution preparation:
NaCl 4,1 g
H20 80 ml
Dissolve
Add slowy, as getting dissolved, 10 g CTAB, heating to 65 C, and
stiring.
Adjust volume to 100 ml.
(CTAB: from Aldrich) CTAB dust is disgusting!

-  Add 150 ul of 5 M NaCl to 600 ml of unpurified DNA solution in
eppendorff tube (from a normal DNA extraction, before phenol or
anything; i.e. after proteinase K-SDS digestion. Also, it can be used
to purify a water solution of DNA purified with other method. I use:
resuspend  1-3 ml of culture of bacterial cells in 560 ul of TE; add 30
ul SDS 10% and 10 ul of proteinase K 20 mg/ml. Incubate at 37 C for 1
hour.). Vortex.
- Add 80 ul CTAB/NaCl. Vortex and heat at 65 C during 10 minutes.
- Add 400 ul chloroform/isoamilic acid 24:1. Vortex and centrifuge in
the epp. 4 minutes.
- Take the supernatant. You can repeat the three steps once more.
- Now, follow a normal phenol/chloroform extraction and EtOH
precipitation. Add RNAse if not done yet. It is very important to
remove all your CTAB from the DNA with the fenol/chlfrm extraction,
because it inhibits certain enzymes. I recommend do phenol extraction
till the interfase is clean.

Good luck! Any questions? Please, post or mail.


----------------------------------
Rafael Maldonado
Howard Hughes Medical Insitute
University of Utah
Rafael at genetics.med.utah.edu



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