Ever thought of utilizing a kit-free DNA gel extraction technique?
Kits for DNA gel extraction are an effective way to save lots of time within the lab, however they’re expensive and might produce plenty of plastic waste. In case you are a PhD scholar, perhaps much more necessary is that utilizing kits stops you from fascinated with what they’re doing and WHY.
You can begin saving cash and decreasing waste by recycling your DNA extraction columns. However this may be a half-measure. Going kit-free can prevent cash, scale back waste, get your mind cells energetic, and allow you to troubleshoot when essential 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 dimension. Then, after visualizing the fragments and evaluating them to a dimension commonplace, you narrow 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. Be aware 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 reduce the dimensions of the fragment. A bigger fragment means extra contamination and extra dilute DNA consequently.
Three Straightforward Package-free DNA Gel Extraction Strategies
Methodology 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 through {an electrical} subject shouldn’t be vertical however horizontal. (1)
On this kit-free technique, you narrow the gel upstream of the working route 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, rigorously 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 recuperate and its quantity, so you could 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 will depend on the dimensions of the fragment, gel density, and voltage. It is advisable standardize situations and will uncover that your fragment has run away from you, and you could repeat the process.
However you’ll be able to keep away from the guesswork of switch time through the use of one of many strategies beneath, which doesn’t require casting a internet within the hope of catching your DNA like a fish.
Methodology 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 dimensions 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 through the spin and collects within the outer tube. Relying on the dimensions of the preliminary gel fragment, the restoration quantity is 15–30 μl.
The authors of the strategy didn’t experiment with fragments bigger than 3 kb. However contemplating that the restoration charge falls as fragment dimension will increase, it’s in all probability not price doing.
Methodology 3: Utilizing Dialysis
Dialysis is one other easy technique 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 accommodates your DNA. It then could be precipitated.
DNA Gel Extraction Strategies Summarized
Desk 1 compares the fee, issue, and restoration for various DNA gel extraction strategies.
Desk 1. Comparability of strategies for kit-free DNA gel extraction
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Downstream ethanol |
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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 ideas, 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 method? Tell us within the feedback beneath!
References
- Lewis, M. DNA extraction from agarose gels (primary technique). MethodBook Accessed 01/06/2022.
- 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.
