Extraction of DNA fragments from gels

Better Crush and Soak, than Crash and Burn!
by Erbay Yigit, Labtimes 03/2011




Antique crush and soak method: the grape gets crushed and the feet are soaked.

Today, the web is a rich source of lab protocols and tips – provided that they are reliable. Here’s an interesting tip on extracting DNA fragments from gels that Lab Times stumbled upon in bioprotocols.info.

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, denatures 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 the expected size. The ­ssDNA may run even slower than dsDNA, if it folds back onto itself and forms a secondary structure.

If the DNA you are using is simple DNA, such as plasmid DNA or a 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 on 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.

If you do not want your DNA to be denatured, I recommend that you use the crush and soak method instead of commercially available kits that use strong chaotropic agents. Although the method takes longer, it yields reliable and high quality DNA.

Crush and Soak Protocol:

  • Using a clean razor blade, excise the slice of agarose/polyacrylamide containing the 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 overnight (48 hours increases recovery).
  • Remove the crush and soak buffer and ethanol precipitate.

Alternatively, to obtain a 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)

(Erbay Yigit is from Turkey and is doing a postdoc in the John Widom lab at the Northwestern University in Illinois, USA)





Last Changed: 10.11.2012




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