Isolation of DNA fragments by the crush and soak method

Isolation of DNA fragments from agarose and polyacrylamide gels by the crush and soak method

There are commercial and non-commercial methods to isolate DNA from agarose and polyacrylamide gels. Perhaps, the most common non-commercial method is the isolation of DNA by the crush and soak method. Although commercially available kits are very convenient and affordable, there are conditions where I personally avoid using these kits.

During the gel extraction of small DNA fragments (~200 bp) with QIAquick Gel Extraction kit (Qiagen), I have noticed that Qiagen Buffer QG, used to melt agarose, denaturs double stranded DNA into single stranded DNA. This is due to Guanidinium thiocyanate, a strong chaotropic agent, present in Buffer QG. The denaturing effect of Buffer QG is worse when DNA fragments are short and AT rich. Moreover, high temperatures and long incubation times increase the denaturing effect of Buffer QG.

Since Ethidium bromide does not bind to single-stranded DNA, it may be difficult to notice ssDNA on a gel. I suggest that you pay attention to the DNA fragments that are shorter than expected size. SsDNA may run even slower than dsDNA if it folds back onto itself and forms a secondary structure.

If DNA you are using is simple DNA such as plasmid DNA or PCR product, it might be possible to re-anneal it. For re-annealing of simple DNA you may want to follow the instructions given by Qiagen at their webpage. However, if the DNA denatured was a complex DNA such as genomic fragments, it is almost impossible to re-anneal the single strands back together. If you do not want your DNA to be denatured, I recommend that you use Crush and Soak Method instead of commercially available kits that use strong chaotropic agents. Although the method takes longer time, it yields reliable and high quality DNA.

Crush and Soak Protocol:

Using a clean razor blade, excise the slice of agarose/polyacrylamide containing DNA fragment.
Place the gel slab into a tube.
Crush it with a teflon pestle into small pieces (freezing the gel slab helps crush better).
Add about 3 volumes (v:v) of crush and soak buffer onto gel pieces.
Shake the tube gently on a rotator over night (48 hours increases recovery).
Remove the crush and soak buffer and ethanol precipitate.
Alternatively, to obtain cleaner product, purify the DNA by a Qiagen QIAquick Column using *Buffer PB*, which does not denature dsDNA.

Crush and Soak Buffer Recipe:

300 mM Sodium Acetate
1 mM EDTA (pH 8.0)
0.1% SDS (optional)

by Erbay Yiğit, 2009.