Questions about DNA recovery by silica binding

Paul N Hengen pnh at ncifcrf.gov
Mon Oct 20 10:41:11 EST 1997


Wilson (netson at uxmail.ust.hk) wrote:

> Thanks for all netters who answer my questions.  I have a few
> questions about the procedure of silica binding.  I have made the silica
> and resuspended in guanidium thiocyanate.  After adding the silica to
> solution which contain dissolved agarose, DNA and guanidium thiocyanate
> salt,  I tap the solution to mix them.  Then should I incubate the mixture
> in ice or at room temp?

Room temperature.

> Which temperature can give better DNA recovery?
> Should I use 50% ethanol with 100mM NaCl, Tris-HCl and EDTA or only 70%
> ethanol as the washing solution?  Finally,  which incubation temperature
> 45, 55 or 65oC can elute the DNA more effective?

I'd use the 50% ETOH wash.  I do the elution at 65 C with H2O, but there was an
article published about the best conditions for recovery depending on the GC
content of your DNA fragment:

@article{Kaur1995,
author = "R. Kaur
     and R. Kumar
     and A. K. Bachhawat",
title = "Selective recovery of {DNA} fragments from
silica particles: effect of {A--T} content and elution
conditions",
journal = "Nucleic Acids Res.",
volume = "23",
pages = "4932-4933",
comment = "A-T rich DNA fragments are poorly recovered",
year = "1995"}

You also might want to read these articles:

@article{Hengen1994Septibs,
author = "P. N. Hengen",
title = "Methods and reagents - Recovering {DNA} from agarose gels",
journal = "Trends in Biochemical Sciences",
volume = "19",
number = "9",
pages = "388-389",
month = "September",
year = "1994"}
Find it at ftp://ftp.ncifcrf.gov/pub/methods/TIBS/sep94.txt

@inproceedings{Hengen1995,
author = "P. N. Hengen",
editor = "A. M. Griffin
     and H. G. Griffin",
booktitle = "Molecular Biology: Current Innovations and Future Trends",
title = "Mini-prep plasmid {DNA} isolation and purification
     using silica-based resins",
publisher = "Horizon Scientific Press",
volume = "Part 1",
address = "Wymondham, U.K.",
pages = "39-50",
year = "1995"}
Look for it on my cv at http://www-lmmb.ncifcrf.gov/~pnh/cv.html

--
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