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3 Simple DNA gel extraction Strategies


Ever thought of utilizing a kit-free DNA gel extraction technique?

Kits for DNA gel extraction are a good way to avoid wasting time within the lab, however they’re expensive and might produce numerous plastic waste. If you’re a PhD scholar, perhaps much more essential is that utilizing kits stops you from excited about what they’re doing and WHY.

You can begin saving cash and lowering waste by recycling your DNA extraction columns. However this might be a half-measure. Going kit-free can prevent cash, scale back waste, get your mind cells lively, and assist you troubleshoot when obligatory or enhance the protocols.

How DNA Extraction Kits Work

Within the case of DNA fragment isolation, there’s an preliminary stage of separating the fragment(s) of curiosity from the remainder of your DNA by working the DNA on a gel that separates them by measurement. Then, after visualizing the fragments and evaluating them to a measurement normal, you chop the band(s) of curiosity, dissolve the agarose and proceed with the extraction.

In a nutshell, the DNA extraction kits work in three steps: bind, wash, and elute. DNA must be extracted from the agarose gel, sure to a substrate, and eluted. Relying on the elution protocol, precipitation and resuspension within the buffer appropriate for downstream functions could also be wanted. Word that PCR normally tolerates much less purified samples than cloning, e.g., you don’t have to reprecipitate your DNA after gel extraction if you happen to use it as a template.

A consideration for all strategies is to attenuate the scale of the fragment. A bigger fragment means extra contamination and extra dilute DNA in consequence.

Three Simple Package-free DNA Gel Extraction Strategies

Technique 1: Bind to Paper

On this technique, 3MM filter paper serves like a nitrocellulose membrane in Southern or northern blot binding DNA, besides the switch by way of {an electrical} discipline isn’t vertical however horizontal. (1) 

On this kit-free technique, you chop the gel upstream of the working path and insert a bit of filter paper, after visualizing your DNA within the gel. Persevering with the electrophoresis binds the fragment to the paper. You’ll then want to clean your bound-to-paper DNA fragment and elute it utilizing a tris-EDTA (TE) buffer or water.

Washing is finished by inserting the paper fragment in a 0.5 ml tube and including 50–100 μl of TE.

Subsequent, fastidiously make a gap within the backside utilizing a syringe needle, place it in a 1.5 ml tube, and spin. The buffer with eluted DNA collects on the backside of the larger tube.

Repeating the elution will increase the quantity of DNA you get well and its quantity, so chances are you’ll have to ethanol precipitate your DNA.

The benefit of this technique is that it doesn’t require a lot, aside from some filter paper and tubes. However the time it takes to bind DNA relies on the scale of the fragment, gel density, and voltage. You should standardize situations and should uncover that your fragment has run away from you, and you’ll want to repeat the process.

However you’ll be able to keep away from the guesswork of switch time by utilizing one of many strategies beneath, which doesn’t require casting a internet within the hope of catching your DNA like a fish.

Technique 2: Utilizing Glass Wool or Cotton Wool

This technique requires two centrifuge tubes—one 0.5 ml and one 1.5 ml—plus some glass wool or cotton wool (simply plain bleached cotton you discover on cotton wool buds). (2)

Punch an inside-to-out gap within the smaller tube and insert it into the larger tube.

Put a few of your chosen wool on the underside of the tube.

Place the fragment on the wool cushion and shut the lid of the smaller tube.

Spin the tube in a microcentrifuge for five–10 minutes, relying on the scale of the fragment:

  • 1 kb: 5,000 rpm for five min
  • 1–2 kb: 10,000 rpm for five min
  • 2–3 kb: 10,000 rpm for 10 min.

The buffer with dissolved DNA filters by means of the wool or cotton throughout the spin and collects within the outer tube. Relying on the scale of the preliminary gel fragment, the restoration quantity is 15–30 μl.

The authors of the tactic didn’t experiment with fragments bigger than 3 kb. However contemplating that the restoration price falls as fragment measurement will increase, it’s in all probability not value doing.

Technique 3: Utilizing Dialysis

Dialysis is one other easy strategy to get your DNA again from a gel. (1)

First, freeze the fragment at –20°C for 30 min.

In the meantime, soak a dry piece of dialysis tubing in distilled water, place it into the identical buffer as your fragment (e.g., TBE), and seal one finish.

Place the fragment into the tubing. Add 200–400 μl of TBE and seal the opposite finish. 

Place into the electrophoresis tank and apply 5V/cm present. The DNA comes out of the gel fragment however stays contained in the tubing. After 10–quarter-hour, unseal one finish of the tubing and take away the buffer inside that comprises your DNA. It then may be precipitated.

DNA Gel Extraction Strategies Summarized

Desk 1 compares the associated fee, problem, and restoration for various DNA gel extraction strategies.

Desk 1. Comparability of strategies for kit-free DNA gel extraction

Downstream ethanol 
precipitation 

For extra assist along with your DNA gel extractions, learn our associated article and get 10 DNA gel extraction tricks to maximize your extractions.

For extra suggestions, methods, and hacks for getting your experiments accomplished, take a look at the Bitesize Bio DIY within the Lab Hub.

Do you employ any of the above DNA gel extraction strategies, or maybe reclaim your gel-bound DNA one other approach? Tell us within the feedback beneath!

References

  1. Lewis, M. DNA extraction from agarose gels (primary technique). MethodBook Accessed 01/06/2022.
  2. Solar, Y, et al. (2012) A fast, cost-free technique of purification of DNA fragments from agarose gel. Journal of Most cancers 3:93–95. 

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