In order to isolate single-strand DNA from M13-based phagemid, we intended to use silica-spin isolation instead of phenol/chloroform extraction for some reasons. Due to unavailability of commercial kits, we have to prepare some buffers by ourselves, but we failed in silica-spin adsorption of ssDNA. Any suggestions for best suited conditions?
I am using a commercially available kit for RNA Isolation to purify tRNA, however this kit is not suited for purification of short RNA. The supplier recommended that instead of using the supplied binding buffer I should add 0.1 volumes of NaOAc pH 5.2 and 3 volumes of isopropanol, the rest of the purification protocol is unchanged. This might work for you as well. If you don't want to "hijack" a commercially available kit, I recommend the following literature:
Boom, R., C. J. Sol, M. M. Salimans, C. L. Jansen, P. M. Wertheim-van
Dillen, and J. van der Noordaa. 1990. Rapid and simple method for purification
of nucleic acids. J. Clin. Microbiol. 28:495–503.
Boom, R., C. Sol, M. Beld, J. Weel, J. Goudsmit, and P. Wertheim-van
Dillen. 1999. Improved silica-guanidiniumthiocyanate DNA isolation procedure
based on selective binding of bovine alpha-casein to silica particles.
J. Clin. Microbiol. 37:615–619.
These publications describe a simple silica-based DNA-isolation method that actually works and you can prepare all buffers yourself.
Good luck!
Best regards,
John
well I had a problem with extraction DNA from biofilms and durind 3 months I tested as many protocols as it was possible.... Nevertheless one of the most important thing was to have pH of my samples almost 7.0 (from 6.6 to 7.0) and used NaOAc pH 7.3 and 0.7 volumes of isopropanol.
Maybe it would help in your reseach as well.
Best,
Tom
Hi all,
check the recipes of QIAGEN buffers at http://openwetware.org/wiki/Qiagen_Buffers
You can prepare your home made solution based on those.
cheers
Thanks to all!
I've choosed after all 4.2M GuHCl/isopropanol buffer for phages lysis and silica spin adsorbtion and obtained ~250 ng/µl pure ssDNA
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