Hi, I have used a lot of kits, and below is one of the best methods for purifying and extracting DNA from gels or solutions. It gives you (i) ultra pure DNA; (2) you can control the elution volume to 5 uL, (3) It is very very affordable. Its based on the GeneClean kit.

Solutions you need:

NaI solution:

6M Sodium Iodide (NaI)

(Wrap in foil to protect from light)

Wash solution:

100 mM NaCl

1mM EDTA (pH8.0)

10 mM Tris.Cl (pH 8.0)

50% Ethanol

(Filter sterilise)

20% w/v Silica milk Slurry:

Weight out 2 g of silica - (sigma S5631)

Add 10 mL of milliQ water. Vortex and incubate on Daisy wheel overnight to make a good suspension.

For Gel Extraction:

1) Weight gel slice in 1.5 mL centrifuge tube, add 3volumes 6M NaI solution

2) Incubate at 45-55 degree for gel to melt agarose

3) Vortex the silica milk slurry to make even suspension

4) add 1-5 uL of silica milk to NaI/DNA solution

5) Spin on daisy wheel (or mix by hand every few minutes) for at least 5 mins, but can be as long as you want

6)Spin tube 5 seconds at max speed to pellet silica/DNA

7) Wash pellet 3X with 500 uL wash buffer, making sure you resuspend silica slurry each time.

8) Spin max speed between washes

9) After last wash, remove the supernatant and dry the silica at room temp for 10 minute- or at 45-55 degree for a few minutes

10) To elute DNA, resuspend the silica in water or TE - the volume you require. For ligations, you may want to resuspend in as little as 10 uL.

11) Wait 1 min, then spin for 30 second to pellet silica

12) Carefully remove supernatant with the eluted DNA into a new tube. Be careful not to take any of the silica with you.

REMEMBER - if you use TBE (borate based gels) - you must add 1/10 volume of 1M sodium phosphate (ph 6.5) and 1M mannitol to a final concentration of 0.1M because borate solution inhibit DNA binding to silica. I just avoid this by always using TAE agarose gels.

Good luck!

Vikas,

To begin with you can go through

http://www.methodbook.net/dna/gelextrc.html

http://www.protocol-online.org/cgi-bin/prot/jump.cgi?ID=3193

Hope that helps.

You can also try "QiaGen gel extraction kit" if available.

I'll also recommend you to use a Kit, e.g. Macherey Nagel oder Quiagen. They are faster (approx. 20 min), have higher yields and are saver than the most non-kit-protocols.

Dear Mr Patel

Plz read the below mentioned protocol in word file. I hope it will work for you.

Best Regards

Best regards

You can put the cut side of the agarose gel on a eppendorff. Add one volume of phenol-chloroform and frozen in liquid nitrogen. Freezing and thawing performed twice and finally centrifuged at RT for 15 minutes. After precipitating the aqueous phase (isopropanol or ethanol) and you're done

When using the product for downstream cloning we use a low-melt agarose. Run the product into low-melt agarose (by excising a block beneath the band you want), excise it, melt it (5mins) then add phenol, vortex thoroughly then cool on ice. Spin at max and transfer the aqueous to another tube. Perform phenol-chloroform now followed by ethanol precipitation - see attached protocol. The quality is good and we find ligations are much more efficient even when the product has low concentrations (we use a nanodrop rather than visualising on a gel) - good luck!

Use Thermo Scientific Gel Extraction Kit....it has always helped me to extract DNA...with good results!

Several techniques can be used to purify DNA from agarose gels, and choosing between them is, to some extent, a matter of personal preference. They all start out by excising the desired "band" from an ethidium-stained gel viewed with a UV transilluminator. Because UV light can fragment DNA, it is best to work expeditiously and keep exposure time to a minumum. Cut out the desired piece of agarose using a razor blade or scalpel blade, and try to get as little extra agarose as possible. The block of agarose containing DNA is then subjected to any of the following.

Electroelution: The block of agarose is placed in a piece of dialysis tubing with a small amount of fresh electrophoresis buffer, the ends sealed with clamps, and the bag placed into an electrophoresis chamber. Application of current will cause the DNA to migrate out of the agarose, but it will be trapped within the bag. Progress can be monitored using a transilluminator, as shown below. When the DNA is out of the agarose, the flow of current is reversed for a few seconds to knock the DNA off of the side of the tubing. The buffer containing the DNA is then collected and the DNA precipitated with ethanol.Electroelution is more time consuming than some of the other techniques, but works well and is probably the best technique for recovery of large (> 5 kb) fragments of DNA.

Binding and elution from glass or silica particles: In an environment of high salt and neutral or low pH, DNA binds avidly to glass, silica gel or diatomaceous earth. This phenomenon can be exploited to purify DNA from solutions containing impurities such as agarose. Typically, a slice of agarose containing the DNA of interest is "melted" by incubation in a solution containing chaotropic salt (e.g. sodium iodide) at a pH of 7.5 or less. Glass powder or silica gel is then added and the suspension is mixed to allow adsorption of DNA. The particles can then be recovered from the original liquid and washed by centrifugation and resuspension in high-salt-ethanol buffer. Finally, the pellet is resuspended in a solution with low or no salt at basic pH, the free particles pelleted by another centrifugation, and the DNA-containing supernate recovered.

The glass or silica particles used for this technique can be prepared in house or, more conveniently, purchased from a number of suppliers.

Like some of the collegues above already mentioned, a lot of kits exist. Anyhow if you want a manual system, here is one. Run your agarose gel (preferably high purity and max 1 % concentration if possible for your fragment size). Stain and cut out as narrow as possible. Fold the agarose slice into a standard 0.2 µm Millipore membrane filter (presaturated with 1 mm EDTA.). Put the filter in an eppendorf tube (pierce a small hole in the bottom with surgical needle beforehand). But this tube on top of another eppendorf. Spin at 3000 rpm in a standard eppendorf centrifuge for 2 minutes. Perform a standard clean up on the filtrate. Yield is lower than kit based extractions but for 99 % of applications this works fine.

It takes some practice but works fine. There are some tricks to avoid UV illumination of your fragment. Ask if needed.

You can also electrophorese the band of interest onto DEAE paper then elute with high salt buffer. There was a protocol in Molecular Cloning (Maniatis) and I think in Current Protocols. There is also a paper online here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC327265/

Try Prepease DNA purification kit from affymetrix (formerly marketed by USB). This is the most easiest and reliable method I have used. I am sure that there are similar products on the market.

http://www.affymetrix.com/catalog/131351/USB/PrepEase-Gel-Extraction-Kit#1_1

Here's what "kit" manufactures don't want you to know: The Trough Method is both the cleanest, cheapest and easiest: Cut a trough into the gel just in front of your band. Fill the trough with polyethylene glycol to trap the DNA. Run the band into the trough. Retrieve with a pipette. Done.

I wrote a blog post about this just a few days ago, with detailed images: http://www.geneandcell.com/blogs/molecular-biology-methods/10883773-dna-purification-from-agarose-gels

One way of possibly major improvement is if you do not use UV at all. If you can afford, use GelSafe, CybrSafe, GelRed or some similar dye for viusualizing the band in blue light like DarkReader.

If you are looking for manual methods over kits, then you may not afford the above mentioned slightly expensive things either, so try running 2 lanes of the DNA and after running it cut the two lanes apart, position the band on one using UV, put them back together and cut the band from the un-exposed lane.

Another trick which is also inexpensive:

Instead of syringe pressure you may use a needle to make a tiny hole on the bottom of a HALF ml eppendorf tube, put a piece of filter paper in the tube to cover the hole and place the excised (possibly freeze-thawed) band on the filter paper. Insert this half ml tube into a 1.5 ml eppendorf tube that will hold it so that it can not slide down to the bottom. Now centrifuge the whole thing so that the buffer come out of the gel slice, through the paper, through the hole into the bigger tube. Done. Precipitate or extract or whatever.

you can use this protocole for DNA extraction from Agarose gel:

Cut DNA-fragments from gel, put in the hand-made 0,5-ml filter tube (with hole), and place the tube in a 1,5-ml tube, store in –20°C for 30 minutes. Centrifuge 5 minutes, 14500 rpm, Set the volume to 100 µl and add 10 µl 2M NaCl, vortex. Add 400 µl 100% EtOH (cold), vortex. Store in –20°C for 15-20 minutes, then centrifuge at 4°C, 15 min., 17500 rpm, Wash with 200 µl 70% EtOH (cold), centrifuge at 4°C, 10 min., 17500 rpm. Dry for 10 min, finally resuspend in 12 µl H2O. Load check on gel (2 µl). Good luck.

I've used so many gel purification kits. The best one so far is MP GeneClean III Kit. It can purify DNA fragments sized from 200 bp to 20 kb, and if you use the optional SPIN filter, you can even purify fragments up to 300 kb. The kit is very simple to use, and the efficiency is very high. This is also the cheapest one in terms of price per prep. For $300 you can do 600 preps. Check out mpbio.com/sampleprep.

Hi,

if you have to simply purify a DNA fragment from agarose gel (do not know wich size it is) I would definetely go for Qiagen purification kit (you can check the fragment size and find the kit that is best for your purposes).

You can use guanidium based salt solution of 5M to 6 M to solubilise the gel and you can move on with silica based spin column method to extract the DNA from your gel.

you can use ready made spin column or you can follow the procedure mentioned by Booms et al in "Rapid and simple method for purification of nucleic acids". J. Clin. Microbiol., 28: 495–503.

You can use the procedure given in Maniatis in which one collects the DNA band in a dialysis bag by electrophoresis and then precipitates the same with ethanol. It can be tedious but now there are a number of kits like Qiagen is available in the market with which one can get about 80% of the DNA from the gel.

Well no one suggested this but one the most common method to purify DNA,is through PEG-NaCl.i did it once and got good results.

Hi Avnika, can u send me a brief protocol for gel purification using PEG- NaCl method?

Here is a solution recipe for DNA gel extraction kit. You can leave the gel in QG solution at 65*C for 5 min or till the gel is completely dissolved. Mix and load the whole thing on any silica membrane based spin columns---> spin and discard the flow though-->wash the column with 600ul of 80% ethanol--->dry the column and elute with 50ul ddH2O (or elute with 5ul ddH2O by using Tini spin column). You should get high quality DNA!

Here is the link to get bulk DNA Mini and Tini spin columns ($36 for 100 columns or $59 for 100 RNA columns):

http://www.enzymax.net/columns/mini_column_DNA.htm

http://www.enzymax.net/columns/tini_spin_DNA_column.htm

QG Buffer (gel solubilization buffer):5.5 M guanidine thiocyanate (GuSCN), 20 mM Tris-HCl, pH 6.6 (25oC), dissolve in pH 7 standard solution (yellow) or water. (Buy bulk buffer from Qiagene cat# 19063, 250 ml)

Good luck!

When you have to get as much DNA as possible from an agarose gel, phenol/chlorophorm extraction is more efficient, but it is more time consuming.

I would suggest you to make a low melting agarose gel 0.6-0.8% in a cold chamber. Then carefully slice your band with UV with a razor blade. Weight your band and complete with water to get 500uL as working volume. Warm at 60ºC until the gel is dissolved, then add NaCl to get 50mM final concentration.

Then extraction phenol (1vol - 500uL), vortex 15-30 sec, centrifuge 5min 13000g RT

Get aqueous phase, extraction with 1vol phenol/chlorophorm/isoamyl alcool (25:25:1), vortex 15-30 sec, centrifuge 5min 13000g RT.

Get aqueous phase, around 400uL, precipitate with 2.5vol EtOH (1mL) 0.1vol NaAc, overnight -20ºC or 1 hour -80ºC.

Centrif. 30min 13000g 4ºC, wash with 70% EtOH 13000g 4ºC, dry and resuspend.

With this protocol much longer that the kit, I get around 80-90% recovery I think.

Optionnal you can add glycoblue as co-precipitant, it is nice as it helps to precipitate and more important you can see the pellet!

Good luck with this!

I cloned several products by cutting the band, adding a small amount of water (20 ul is ok), freezing in -80 for 5-10 minutes and centrifuge for 1 min full speed (13000 rpm). Take the el eluted water and use it for cloning. I hope it helps

An alternative method to the syringe it to freeze squeeze the gel. After cutting out the band. Freeze the piece of agartose gel. After freezing it to -20oC take the piece out of the freezer and squeeze the liquid out of the gel between parafilm. Pipet the liquid in an eppendorp tube and do an ethanolo or propanol precipitati or use it directly for cloning if the concentration is high enough to overcome buffer differences.

I am currently doing cloning experiments and also needed to purify from agarose gel.

What I do is the following: after my PCR I visualize on gel. If i have a very strong specific amplicon band, I clean up the leftover PCR product using a macherey nagel robot we have in our lab. If I have a very strong specific amplicon band, together with some other non specific bands (which are a lot less intense), I do the exact same thing. After cloning, 99% of my clones has the correct insert!

However, if I have several bands on gel that are more or less the same intensity as my specific amplicon, I cut out the band from agarose gel and place it in 30µl of MQ or 1x TE for 3 days (in the fridge). The DNA simply migrates to the MQ or buffer. Afterwards, I use this eluens in a new PCR, which gives me one specific band after visualization. That PCR product is cleaned with the Macherey Nagel robot.

You can also use the eluens directly for the ligation step if you clean it with the Macherey Nagel system, or probably even without cleaning it.

Which method do you use for the quantification of your DNA? If you use Nanodrop, I'd say don't trust the results you get. I made the comparison between several methods and found out only the Qubit gives nearly correct results.

And even if I only have a cleaned amplicon product with a concentration of 3 ng/µl, the ligation still works fine.

I also tested several gel extraction kits, and what I can say is that they cost a lot of money for bad results. They promise 95% recovery, but none of them do. By testing some things by yourself, you can get around expensive kits and find a method that is much faster and cheaper.

For gel extraction, I also tested about 5-10 methods I thought about myself and in the end the most simple methods work most of the time.

Cut DNA along with gel and do reverse electrophoresis and collect DNA and precipitate with ethanol.

I advice you to the Gel Exctraction Kit "Qiaquick", it is a simple and fast to use, and gives a good yield. This is the kit we use in our laboratory and it's marketed by Qiagen.

This method from the web works well, but for any of the freezing methods I would recommend freezing below -20°C. The colder you freeze, the easier it is to get rid of the agarose

DNA Purification - Freezing Method

Jeff Lawrence

1. Remove gel slice contain DNA fragment and place in 10 volumes of:

300 mM NaOAc, pH 7.0 (300 mL 1 M NaOAC, pH 7.0)

1 mM EDTA (2 mL 500 mM EDTA, pH 8.0)

(698 mL ddH20)

2. Incubate at 22° for 30 min. Transfer gel slice to a fresh tube.

3. Place tube in a Dry Ice/Ethanol bath for 5 min.

4. Puncture the bottom of a 0.5 mL microcentrifuge tube with a needle. Place the gel slice into this tube. Place this tube inside a 1.5 mL microcentrifuge tube.

5. Centrifuge for 15 min.

6. Collect the Eluent from the 1.5 mL eppendorf tube. Extract and precipitate the DNA.

I used electroelution. Quite good yield and it also does not need too much time. You can just google "electroelution" to find it.

Hi, I have used a lot of kits, and below is one of the best methods for purifying and extracting DNA from gels or solutions. It gives you (i) ultra pure DNA; (2) you can control the elution volume to 5 uL, (3) It is very very affordable. Its based on the GeneClean kit.

Solutions you need:

NaI solution:

6M Sodium Iodide (NaI)

(Wrap in foil to protect from light)

Wash solution:

100 mM NaCl

1mM EDTA (pH8.0)

10 mM Tris.Cl (pH 8.0)

50% Ethanol

(Filter sterilise)

20% w/v Silica milk Slurry:

Weight out 2 g of silica - (sigma S5631)

Add 10 mL of milliQ water. Vortex and incubate on Daisy wheel overnight to make a good suspension.

For Gel Extraction:

1) Weight gel slice in 1.5 mL centrifuge tube, add 3volumes 6M NaI solution

2) Incubate at 45-55 degree for gel to melt agarose

3) Vortex the silica milk slurry to make even suspension

4) add 1-5 uL of silica milk to NaI/DNA solution

5) Spin on daisy wheel (or mix by hand every few minutes) for at least 5 mins, but can be as long as you want

6)Spin tube 5 seconds at max speed to pellet silica/DNA

7) Wash pellet 3X with 500 uL wash buffer, making sure you resuspend silica slurry each time.

8) Spin max speed between washes

9) After last wash, remove the supernatant and dry the silica at room temp for 10 minute- or at 45-55 degree for a few minutes

10) To elute DNA, resuspend the silica in water or TE - the volume you require. For ligations, you may want to resuspend in as little as 10 uL.

11) Wait 1 min, then spin for 30 second to pellet silica

12) Carefully remove supernatant with the eluted DNA into a new tube. Be careful not to take any of the silica with you.

REMEMBER - if you use TBE (borate based gels) - you must add 1/10 volume of 1M sodium phosphate (ph 6.5) and 1M mannitol to a final concentration of 0.1M because borate solution inhibit DNA binding to silica. I just avoid this by always using TAE agarose gels.

Good luck!

I highly recommend MP Biomedical's GeneClean Kit. You can purify DNA from 200 bp to 20 kb in size and the yield is excellent. It takes only about 20 min, much less time consuming than other products. For about $300 kit, you can do 600 preps. Good luck.

I agree with Rongfeng, the protocol I gave is based on the gene clean kit. 

thanks to all of you

Recovery of DNA from LMP (Low Melting Point) Agarose Gel

1. Separate DNA fragments through an LMP agarose gel containing ethidium bromide (0.5 microgram/ml).

2. Detect DNA by irradiating the gel with long-wave ultraviolet light (wave length: 355 - 360 nm).

3. Cut out the region of the gel containing desired DNA. Transfer the gel slice into a microfuge tube.

4. Add to the gel slice about 1-3 volumes of TE. Add 0.3 volume of 3M sodium acetate (pH7).

5. Heat the tube at 65 C. Eventually tap the tube to mix the melted gel and TE.

6. Add about equal volume of phenol saturated with TE. Vortex mix for 30 seconds.

7. Centrifuge for 2 min at room temperature, then for 5 min at 4 C.

8. Transfer the aquaous phase into new tube.

9. Add about equal volume of TE-saturated phenol. Vortex for 30 seconds.

10. Centrifuge for 5 min at 4 C.

11. Transfer the aquaous phase into new tube.

12. Extract the solution with an equal volume of ethylether to remove trace amount of phenol dissolve in the DNA solution.

13. Concentrate DNA with ethanol precipitation.

I have used the protocol described by Charles E. De Bock. It's working absolutely fine and a good yield too.

Thanks for the feedback Ajit. Glad its working!

hello Dr charles...is there any alternative to daisy wheel 

Hi Mayank - the daisy wheel is more for convenience so you can walk away. The binding will occur within the first 5 minutes - but you can leave over lunch if you needed to for example. If you do not have a daisy wheel or other rotating device, you could use a shaker.vortex (on low) to agitate/mix the DNA/silica slurry. If you have none of these, you could just pipette up and down 3-4X every minute for a few minutes whilst it sits on your lab bench. Hope this helps.

I use Thermo Fisher's Gel Extraction kit. High yield, high purity and so easy to use. Check their web site. The catalog number is K0692

Hi Everyone, just to make it clear about the wash solution because some people have been asking about how to make it.

First make three stock solutions of 5M NaCl, 0.5M EDTA and 1M Tris pH 8.0.

1) The easiest was to begin is to buy a 0.5 M EDTA solution pH 8.0 (e.g. https://sciencing.com/make-edta-solution-6452756.html)

Or you can make it using the following instructions: https://sciencing.com/make-edta-solution-6452756.html

2) For Tris Cl. pH80: First make a 1M stock of Tris base - weight out 121.14 g (molecular weight = 121.14) and add milliQ or distilled water to 900 mL. Adjust pH (carefully!!) with HCl until pH = 8.0. Note - if you go below 8.0 you need to start again. DO NOT add NaOH to increase the pH.

You can also buy 1M Tris pH 8.0 as well which can be very convenient.

3) For NaCl - make a 5M stock solution - weigh our 292.2 g into 1 L of milliQ or DI water.

Now lets make a 10X Wash solution from these three solutions which will be 1M NaCl,

10mM EDTA (pH8.0), 100 mM Tris.Cl (pH 8.0)

Take 200 mL of 5M NaCl, 100 mL of 1M Tris Cl, 20 mL of the 0.5M EDTA - and make up to 1L with milliQ or Distilled water. Label this as 10X wash solution and keep it at 4 degrees.

Finally, you want to make your working 1X solution: , I would make 200 mL. Take 20 mL of your 10X stock, add 80 mL of milliQ or distilled water, and then add 100 mL of 100% ethanol. Keep this at -20 degrees. It will keep for a few months.