hi Amir

you can try one of the following methods, which ever eases you

Isolation of DNA from Agarose Gels (Paper Slurry Method)

This procedure isolates DNA from agarose gels by filtration through a filter-paper column. The column is made in a 500 µL tube from a slurry of filter paper in TE buffer.

Materials

• Whatman 3MM filter paper: 50 cm2 piece

• TE Buffer (10X): dissolve 186 mg EDTA and 605 mg Tris in 50 mL dH2O; pH to 8.0 with HCl. Store at 4°C, dilute ten times before using.

• Paper Slurry: cut 50 cm2 of filter paper into tiny (1-2 mm2) pieces; add to 40 mL of TE buffer in a 50 mL tube. Shake vigorously by hand for at least 5 minutes. Store at 4°C.

• Agarose gel with DNA to be isolated

• Clean razor blade

• Filter column: Punch a small hole in the bottom of a 500 µL tube with a 23 gauge needle. Remove paper slurry piecewise with tweezers and place over the hole in the 500 µL tube. Pack with a 200 µL pipet tip and remove excess liquid. Repeat until paper column is approximately 3 mm high. Place inside a 1.5 mL tube to collect eluent.

Procedure

1. Excise the DNA band from the surrounding gel with a clean razor blade; be sure to remove as little gel as possible.

2. Dice the excised gel fragment into small pieces with the same razor; transfer onto filter column.

3. Centrifuge for 10 minutes at highest speed (approximately 20,000g) in a microcentrifuge.

4. Transfer eluent to a fresh tube; recentrifuge agarose

5. Combine eluent with that from previous centrifugation

6. Assemble a second spin column and transfer remaining agarose to the new column. Spin again.

7. Combine all eluent fractions and concentrate via cold ethanol precipitation

or you can use this protocol

Gene-Kleen protocol

Purification of DNA from agarose gels is an essential method involved in the sub-cloning of DNA fragments. The following method describes a variation of the method of Vogelstein and Gillespie, 1979 (Proc. Natl. Acad. Sci. USA. 76, (2) 615-619).

1) Excise the band of interest from the TAE gel using a clean scalpel blade and place in a pre-weighed eppendorf tube.

2) Add 3 volumes of 6M NaI, 0.1% sodium thiosulphate solution and allow agarose to melt (approx. 5 minutes with vortexing). For TBE gels, 0.1 volumes of 1M mannitol should also be added to aid gel solubilisation.

3) Vortex glass suspension (finely crushed glass scintillation vial suspended 1:1 in sterile nanopure H2O or Fluka analytical filter aid resuspended 1:4 in sterile nanopure H2O) and add 7.5ul to agarose solution (7.5ul should be used for up to 5ug DNA and thereafter an extra 1ul should be used for each extra ug DNA).

4) Allow DNA to bind for 15-20 minutes at room temperature.

5) Spin down glass-DNA for 30 secs. in a microfuge at maximum speed and carefully remove supernatant. Discard supernatant.

6) Wash pellet in 500ul 4.5M NaI, 0.1% sodium thiosulphate, re-pellet and discard supernatant.

7) Wash pellet 2 x 500ul in 1 x TE, pH 7.5, 200mM NaCl, 60% EtOH. After last centrifugation, remove all trace of the supernatant and allow to air-dry for 5 minutes.

8) Elute DNA at 37 - 55°C in 25ul TE, pH 8.0 for 5 minutes.

N.B: It is important to elute in a buffer with a pH of ca. 8 as elution in water or lower pH buffers decreases the yield markedly.

9) Spin down glass for 5 minutes at maximum speed in a microfuge and carefully remove supernatant to a clean eppendorf tube.

10) Repeat elution step and pool supernatants. Discard pellet. Respin pooled sample to remove traces of glass for 5 mins and transfer clean supernatant to a fresh, sterile eppendorf tube.

All reagents are made from the highest grade chemicals available (esp. important for the NaI). NaI solutions were made with sterile, nano-pure H2O and final ethanol was made minus ethanol, autoclaved and ethanol added to a final concentration o f 70% (v/v) after sterilisation. Glass 'beads' were made from a finely crushed scintillation vial (i.e high quality glass) by crushing with a pestle and mortar. Glass is crushed basically until your wrist feels like it's about to fall off...and then some (should behave like cooking flour). Alternatively, I have recently tried Celite Analytical Filter Aid (Celite AFA, Cat. No. 22137 - I think thats the catalogue number anyhow!) from Fluka in place of the crushed glass with brilliant results.

you may use the below protocol

Elution of DNA fragments from agarose

DNA fragments are eluted from low-melting temperature agarose gels using an unpublished procedure first developed by Dr. Roe. Here, the band of interest is excised with a sterile razor blade, placed in a microcentrifuge tube, frozen at -70degC, and then melted. Then, TE-saturated phenol is added to the melted gel slice, and the mixture again is frozen and then thawed. After this second thawing, the tube is centrifuged and the aqueous layer removed to a new tube. Residual phenol is removed with two ether extractions, and the DNA is concentrated by ethanol precipitation.

Protocol

1. Place excised DNA-containing agarose gel slice in a 1.5 ml microcentrifuge tube and freeze at -70degC for at least 15 minutes, or until frozen. It is possible to pause at this stage in the elution procedure and leave the gel slice frozen at -70degC.

2. Melt the slice by incubating the tube at 65degC.

3. Add one-volume of TE-saturated phenol, vortex for 30 seconds, and freeze the sample at -70degC for 15 minutes.

4. Thaw the sample, and centrifuge in a microcentrifuge at 12,000 rpm for 5 minutes at room temperature to separate the phases. The aqueous phase then is removed to a clean tube, extracted twice with equal volume ether, ethanol precipitated, and the DNA pellet is rinsed and dried.

you may use the below protocol

DNA Purification - Freezing Method

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.

you may use the below protocol

Another Method for Elution of DNA fragments from agarose

DNA fragments are eluted from low-melting temperature agarose gels using an unpublished procedure first developed by Dr. Roe. Here, the band of interest is excised with a sterile razor blade, placed in a microcentrifuge tube, frozen at -70degC, and then melted. Then, TE-saturated phenol is added to the melted gel slice, and the mixture again is frozen and then thawed. After this second thawing, the tube is centrifuged and the aqueous layer removed to a new tube. Residual phenol is removed with two ether extractions, and the DNA is concentrated by ethanol precipitation.

Protocol

1. Place excised DNA-containing agarose gel slice in a 1.5 ml microcentrifuge tube and freeze at -70degC for at least 15 minutes, or until frozen. It is possible to pause at this stage in the elution procedure and leave the gel slice frozen at -70degC.

2. Melt the slice by incubating the tube at 65degC.

3. Add one-volume of TE-saturated phenol, vortex for 30 seconds, and freeze the sample at -70degC for 15 minutes.

4. Thaw the sample, and centrifuge in a microcentrifuge at 12,000 rpm for 5 minutes at room temperature to separate the phases. The aqueous phase then is removed to a clean tube, extracted twice with equal volume ether, ethanol precipitated, and the DNA pellet is rinsed and dried.

you may use the below protocol

DNA Fragment Purification from Agarose or Acrylamide

For fragments from 200 bp to 10 kb the agarose purification is ideal. For smaller fragments (20 bp to 400 bp) the acrylamide purification is preferred.

Solutions

Crush and Soak Solution

500 mM NH4OAc 3.3 g NH4OAc

0.1% SDS 0.1 g SDS

0.1 mM EDTA 20 ml 500 mM EDTA

up to 100 ml with Q

store at room temperature

3 M NaOAc pH 5.2

24.6 g anhydrous sodium acetate

pH to 5.2 with acetic acid and bring up to 100 ml with Q

store at room temperature

Other Reagents

DMCS treated glass wool (Alltech Assoc. Inc. #4037, 50 g)

0.22 mm disposable micro tip filters (syringe type)

blue tips with melted tips to serve as pestle for crushing acrylamide

Procedure

agarose gels

• Prepare spin columns by cutting off the cap of a 0.5 ml eppendorf tube and forming a hole in the bottom with a hot 18 ga needle. Fill this "mini-column" with a small ball of DMCS treated glass wool and pack down with a pipet tip.

• Cut out the desired band from an agarose gel and place in a spin column inside a 1.5 ml eppendorf tube with the top cut off.

• Spin at 6,000 rpm in a microfuge for 10 minutes.

• Phenol/chloroform extract the flow through and EtOH precipitate with glycogen or tRNA and 10% v/v of 3M NaOAc pH 5.2.

• Wash and dry, resuspend in 20 microliters TE, run 10 microliters on a gel and use 1-2 microliters for a ligation.

acrylamide

• Run a 4-6% acrylamide gel in 1X TBE, stain in EthBr (1-10 mg/ml) and cut out the desired band.

• Crush the acrylamide with a p1000 tip with a melted end to resemble a pestle for the eppendorf "mortar."

• Add 1 ml crush and soak solution and incubate overnight at 37° C.

• Spin in the microfuge for 10 minutes at 14,000 rpm. Remove as much liquid as possible and add another 500 microliters of crush and soak solution.

• Repeat the spin and pool the recovered supernatant.

• Add 0.1 volume of 3M NaOAc, 2.5 volumes of EtOH and carrier (see above).

• Spin as usual, wash and dry. Resuspend in 20 microliters TE.

you may use the below protocol

Rapid elution of DNA from agarose gels

A method of quickly purifying agarose gel DNA fragments for use in subsequent reactions such as further restriction enzyme modification, T4 ligase, Taq polymerase, Klenow, or polynucleotide kinase. Although higher yields of purified DNA can be obtained from commercially available purification kits, greater ligation efficiencies per given amount of DNA have been seen with the use of these described spin columns.

Materials

650 microliter Eppendorf tubes

1.7 ml Eppendorf tubes

Sterile silanized glass wool

Hypodermic needle and syringe

Glass wool spin column preparation

1. Using a clean hypodermic needle, drive a hole in the end of a 650 microliter Eppendorf tube from the inside then cut off the cap.

2. Wearing gloves, pull off a small piece of glass wool and roll it into a ball between hands. Place the glass wool plug in the bottom of the 650 microliter tube. You may need to use a 1ml pipet tip to pack the glass wool in the bottom of the tube. Do not cut the glass wool, as this may produce small glass fibers that can pass through the hole in the small tube.

3. Place the tube with the glass wool in a 1.7ml Eppendorf tube. This completed spin column is now ready for use.

Eluting DNA from agarose gel fragments

1. Visualize ethidium bromide stained agarose gel with a transilluminator on low setting. Working quickly to minimize UV exposure to the DNA, excise the fragment of interest with a clean razor blade. After removing excess liquid with a Kimwipe, place the agarose fragment in the spin column.

2. Centrifuge the tube at 2874 xg for no more than 45 seconds to elute the DNA. Spinning longer coelutes substances that are inhibitory to further enzymatic reactions. 2874 xg corresponds to 5500rpm for Eppendorf centrifuge model 5415; 5000rpm for model 5417.

3. Using a transilluminator, check eluent in bottom of outer tube for the presence of ethidium bromide stained DNA.

4. The eluted DNA can now be used directly in enzymatic reactions. For ligations, no more than 40% of the total reaction volume should be eluted DNA, as this reduces ligation efficiencies.

5. The DNA fragments can be further purified by ethanol precipitation. The addition of 2-4 micrograms of carrier tRNA prior to precipitation can increase yield, and its presence in ligation reactions is non-inhibitory.

or you may use the below protocol

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.

Best Regards

Sibtain

Methodologies vary depending on amount of DNA and downstream application. Can you provide some more details about your system?

Do you mean DNA extraction from agarose gels?

I have used this method, which is based on that from Methods in Molecular and Cellular Biology 3:190-192 (1992):

-Slice out the fragment of interest and cut into 1-2 mm pieces.

-Place the agarose pieces into an Ependorff tube and add an equal volume of phenol, allowing them to mix.

-Place the tube in the ultrafreezer (-80°C)for 5 min, at 20°C for 20 min, or into liquid nitrogen for some seconds, so it freezes completely.

-Centrifuge at 12,000 rpm for 15 min at room temperature.

-Recover supernatant and precipitate DNA by addition of 1/10 vol of NaCl 5M and 2.5 vol of cold absolute ethanol.

-Place at -80°C for 15 min or -20°C for 30 min.

-Centrifuge at 12,000 rpm for 5 min, wash the pellet with 70% ethanol, dry out and resuspend in water or TE.

I have used the purified fragments for ligations without a problem.

You can also check out these alternatives:

http://link.springer.com/protocol/10.1385%2F1-59259-409-3%3A137

Thank you. I want to extract a double digested 7000bp plasmid approximately 3 ug from agarose gel 1% to use it in ligation.

what's wrong with kits then?

In my experience, Qiagen's column kits have poor yield for gel extracted plasmids >6kb in size (although I would expect some success with 3 ug of starting material). QiaEx II beads work better in my experience, and allow you to elute in a smaller volume. I've also heard good things about yields from the Zymoclean gel DNA kits.

This is an archaic method which I haven't tried for years (I'm a total QIAGEN convert!). Take a 0.5ml eppendorf tube, make a pin hole in the bottom, put a plug of glass wool in the bottom, then add your agarose slice. Put this doctored tube into a 1.5ml tube, then centrifuge. Liquid (and DNA) should come out of the gel, through the wool and the pin hole and into the larger tube. You can then ethanol precipitate it to get rid of any salts as mentioned above by Gustavo.

I use OMEGA's kit and it works well in my experience.

Claire this is the TIBURON (SHARK) METHOD, prehistoric but i have sequenced DNA with this method.

Used method used by Claire, it work's

Thanks Lazaro, didn't realise the method actually had a name! :)

I use the dialysis tube like this (Cellu･Sep T2 MWCO：6,000～8,000).

First, agarose gel fragment containing DNA fragment is put in the tube with TAE buffer.

Second, I do the electrophoresis for about an hour, then, the DNA fragment is pulled out into TAE buffer.

Third, the DNA fragment is removed by ethanol precipitation.

This method can collect very large size of DNA fragment more than 100kbp.

We also had in our lab some problems with QIAGEN kits. Recently we switched to AnaliticJena-Biometra kits. In my former lab I was working with QiaEx II and I confirm that it seems to work better than standard Qiagen Kit.

I have tried Claire's method, but substitute the glass wool with filter floss wool for aquarium filters as it is less hassle to use, does not bind DNA in the way that glass does and a large bag will last a lab of 10 people 3 year and is really cheap. Check your recovery by quickly looking at the wool/gel mix under UV and add more buffer and repeat as needed.

If the DNA is intermediate in size (say 2-7kb), cut out the slice of the gel and put it in a zip-lock bag in the freezer. When it is frozen, squeeze the plastic between your fingers and collect the liquid in a pipette tip. Ethanol precipitate.

Cut out the band size of interest from the gel and insert it into an eppendorf tube containing 50ulor less TE buffer. keep the suspension at 4 or 20C overnight until required for use.

Hi Amir,

You can try following the protocol mentioned below:

Use Low melting agarose, cut out you band, add ~5 times vol. Low melting temp elution buffer (20mM Tris, 1mM EDTA ph8.0). Heat for 2-5 mins, with frequent votexing. Add Tris- saturated Phenol, centrifuge at 4000g, 10mins.Extract aqueous layer and repeat the same once with P:C and once with C:I.Add 2 volumes of chilled Abs. Alcohol and 10M ammonium acetate. You can keep this overnight at 4 degrees for better precipitation or can further centrifuge at 10000rpm and proceed to 70 percent alcohol wash. Dissolve pellet in TE ph 8.0

This protocol has been taken from Molecular Cloning by Sambrook. I use this and the efficiency of DNA yeild is decent enough. I use Sigma's Low melting agarose for the same.

All the best :)

hi Amir

you can try one of the following methods, which ever eases you

Isolation of DNA from Agarose Gels (Paper Slurry Method)

This procedure isolates DNA from agarose gels by filtration through a filter-paper column. The column is made in a 500 µL tube from a slurry of filter paper in TE buffer.

Materials

• Whatman 3MM filter paper: 50 cm2 piece

• TE Buffer (10X): dissolve 186 mg EDTA and 605 mg Tris in 50 mL dH2O; pH to 8.0 with HCl. Store at 4°C, dilute ten times before using.

• Paper Slurry: cut 50 cm2 of filter paper into tiny (1-2 mm2) pieces; add to 40 mL of TE buffer in a 50 mL tube. Shake vigorously by hand for at least 5 minutes. Store at 4°C.

• Agarose gel with DNA to be isolated

• Clean razor blade

• Filter column: Punch a small hole in the bottom of a 500 µL tube with a 23 gauge needle. Remove paper slurry piecewise with tweezers and place over the hole in the 500 µL tube. Pack with a 200 µL pipet tip and remove excess liquid. Repeat until paper column is approximately 3 mm high. Place inside a 1.5 mL tube to collect eluent.

Procedure

1. Excise the DNA band from the surrounding gel with a clean razor blade; be sure to remove as little gel as possible.

2. Dice the excised gel fragment into small pieces with the same razor; transfer onto filter column.

3. Centrifuge for 10 minutes at highest speed (approximately 20,000g) in a microcentrifuge.

4. Transfer eluent to a fresh tube; recentrifuge agarose

5. Combine eluent with that from previous centrifugation

6. Assemble a second spin column and transfer remaining agarose to the new column. Spin again.

7. Combine all eluent fractions and concentrate via cold ethanol precipitation

or you can use this protocol

Gene-Kleen protocol

Purification of DNA from agarose gels is an essential method involved in the sub-cloning of DNA fragments. The following method describes a variation of the method of Vogelstein and Gillespie, 1979 (Proc. Natl. Acad. Sci. USA. 76, (2) 615-619).

1) Excise the band of interest from the TAE gel using a clean scalpel blade and place in a pre-weighed eppendorf tube.

2) Add 3 volumes of 6M NaI, 0.1% sodium thiosulphate solution and allow agarose to melt (approx. 5 minutes with vortexing). For TBE gels, 0.1 volumes of 1M mannitol should also be added to aid gel solubilisation.

3) Vortex glass suspension (finely crushed glass scintillation vial suspended 1:1 in sterile nanopure H2O or Fluka analytical filter aid resuspended 1:4 in sterile nanopure H2O) and add 7.5ul to agarose solution (7.5ul should be used for up to 5ug DNA and thereafter an extra 1ul should be used for each extra ug DNA).

4) Allow DNA to bind for 15-20 minutes at room temperature.

5) Spin down glass-DNA for 30 secs. in a microfuge at maximum speed and carefully remove supernatant. Discard supernatant.

6) Wash pellet in 500ul 4.5M NaI, 0.1% sodium thiosulphate, re-pellet and discard supernatant.

7) Wash pellet 2 x 500ul in 1 x TE, pH 7.5, 200mM NaCl, 60% EtOH. After last centrifugation, remove all trace of the supernatant and allow to air-dry for 5 minutes.

8) Elute DNA at 37 - 55°C in 25ul TE, pH 8.0 for 5 minutes.

N.B: It is important to elute in a buffer with a pH of ca. 8 as elution in water or lower pH buffers decreases the yield markedly.

9) Spin down glass for 5 minutes at maximum speed in a microfuge and carefully remove supernatant to a clean eppendorf tube.

10) Repeat elution step and pool supernatants. Discard pellet. Respin pooled sample to remove traces of glass for 5 mins and transfer clean supernatant to a fresh, sterile eppendorf tube.

All reagents are made from the highest grade chemicals available (esp. important for the NaI). NaI solutions were made with sterile, nano-pure H2O and final ethanol was made minus ethanol, autoclaved and ethanol added to a final concentration o f 70% (v/v) after sterilisation. Glass 'beads' were made from a finely crushed scintillation vial (i.e high quality glass) by crushing with a pestle and mortar. Glass is crushed basically until your wrist feels like it's about to fall off...and then some (should behave like cooking flour). Alternatively, I have recently tried Celite Analytical Filter Aid (Celite AFA, Cat. No. 22137 - I think thats the catalogue number anyhow!) from Fluka in place of the crushed glass with brilliant results.

you may use the below protocol

Elution of DNA fragments from agarose

DNA fragments are eluted from low-melting temperature agarose gels using an unpublished procedure first developed by Dr. Roe. Here, the band of interest is excised with a sterile razor blade, placed in a microcentrifuge tube, frozen at -70degC, and then melted. Then, TE-saturated phenol is added to the melted gel slice, and the mixture again is frozen and then thawed. After this second thawing, the tube is centrifuged and the aqueous layer removed to a new tube. Residual phenol is removed with two ether extractions, and the DNA is concentrated by ethanol precipitation.

Protocol

1. Place excised DNA-containing agarose gel slice in a 1.5 ml microcentrifuge tube and freeze at -70degC for at least 15 minutes, or until frozen. It is possible to pause at this stage in the elution procedure and leave the gel slice frozen at -70degC.

2. Melt the slice by incubating the tube at 65degC.

3. Add one-volume of TE-saturated phenol, vortex for 30 seconds, and freeze the sample at -70degC for 15 minutes.

4. Thaw the sample, and centrifuge in a microcentrifuge at 12,000 rpm for 5 minutes at room temperature to separate the phases. The aqueous phase then is removed to a clean tube, extracted twice with equal volume ether, ethanol precipitated, and the DNA pellet is rinsed and dried.

you may use the below protocol

DNA Purification - Freezing Method

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.

you may use the below protocol

Another Method for Elution of DNA fragments from agarose

DNA fragments are eluted from low-melting temperature agarose gels using an unpublished procedure first developed by Dr. Roe. Here, the band of interest is excised with a sterile razor blade, placed in a microcentrifuge tube, frozen at -70degC, and then melted. Then, TE-saturated phenol is added to the melted gel slice, and the mixture again is frozen and then thawed. After this second thawing, the tube is centrifuged and the aqueous layer removed to a new tube. Residual phenol is removed with two ether extractions, and the DNA is concentrated by ethanol precipitation.

Protocol

1. Place excised DNA-containing agarose gel slice in a 1.5 ml microcentrifuge tube and freeze at -70degC for at least 15 minutes, or until frozen. It is possible to pause at this stage in the elution procedure and leave the gel slice frozen at -70degC.

2. Melt the slice by incubating the tube at 65degC.

3. Add one-volume of TE-saturated phenol, vortex for 30 seconds, and freeze the sample at -70degC for 15 minutes.

4. Thaw the sample, and centrifuge in a microcentrifuge at 12,000 rpm for 5 minutes at room temperature to separate the phases. The aqueous phase then is removed to a clean tube, extracted twice with equal volume ether, ethanol precipitated, and the DNA pellet is rinsed and dried.

you may use the below protocol

DNA Fragment Purification from Agarose or Acrylamide

For fragments from 200 bp to 10 kb the agarose purification is ideal. For smaller fragments (20 bp to 400 bp) the acrylamide purification is preferred.

Solutions

Crush and Soak Solution

500 mM NH4OAc 3.3 g NH4OAc

0.1% SDS 0.1 g SDS

0.1 mM EDTA 20 ml 500 mM EDTA

up to 100 ml with Q

store at room temperature

3 M NaOAc pH 5.2

24.6 g anhydrous sodium acetate

pH to 5.2 with acetic acid and bring up to 100 ml with Q

store at room temperature

Other Reagents

DMCS treated glass wool (Alltech Assoc. Inc. #4037, 50 g)

0.22 mm disposable micro tip filters (syringe type)

blue tips with melted tips to serve as pestle for crushing acrylamide

Procedure

agarose gels

• Prepare spin columns by cutting off the cap of a 0.5 ml eppendorf tube and forming a hole in the bottom with a hot 18 ga needle. Fill this "mini-column" with a small ball of DMCS treated glass wool and pack down with a pipet tip.

• Cut out the desired band from an agarose gel and place in a spin column inside a 1.5 ml eppendorf tube with the top cut off.

• Spin at 6,000 rpm in a microfuge for 10 minutes.

• Phenol/chloroform extract the flow through and EtOH precipitate with glycogen or tRNA and 10% v/v of 3M NaOAc pH 5.2.

• Wash and dry, resuspend in 20 microliters TE, run 10 microliters on a gel and use 1-2 microliters for a ligation.

acrylamide

• Run a 4-6% acrylamide gel in 1X TBE, stain in EthBr (1-10 mg/ml) and cut out the desired band.

• Crush the acrylamide with a p1000 tip with a melted end to resemble a pestle for the eppendorf "mortar."

• Add 1 ml crush and soak solution and incubate overnight at 37° C.

• Spin in the microfuge for 10 minutes at 14,000 rpm. Remove as much liquid as possible and add another 500 microliters of crush and soak solution.

• Repeat the spin and pool the recovered supernatant.

• Add 0.1 volume of 3M NaOAc, 2.5 volumes of EtOH and carrier (see above).

• Spin as usual, wash and dry. Resuspend in 20 microliters TE.

you may use the below protocol

Rapid elution of DNA from agarose gels

A method of quickly purifying agarose gel DNA fragments for use in subsequent reactions such as further restriction enzyme modification, T4 ligase, Taq polymerase, Klenow, or polynucleotide kinase. Although higher yields of purified DNA can be obtained from commercially available purification kits, greater ligation efficiencies per given amount of DNA have been seen with the use of these described spin columns.

Materials

650 microliter Eppendorf tubes

1.7 ml Eppendorf tubes

Sterile silanized glass wool

Hypodermic needle and syringe

Glass wool spin column preparation

1. Using a clean hypodermic needle, drive a hole in the end of a 650 microliter Eppendorf tube from the inside then cut off the cap.

2. Wearing gloves, pull off a small piece of glass wool and roll it into a ball between hands. Place the glass wool plug in the bottom of the 650 microliter tube. You may need to use a 1ml pipet tip to pack the glass wool in the bottom of the tube. Do not cut the glass wool, as this may produce small glass fibers that can pass through the hole in the small tube.

3. Place the tube with the glass wool in a 1.7ml Eppendorf tube. This completed spin column is now ready for use.

Eluting DNA from agarose gel fragments

1. Visualize ethidium bromide stained agarose gel with a transilluminator on low setting. Working quickly to minimize UV exposure to the DNA, excise the fragment of interest with a clean razor blade. After removing excess liquid with a Kimwipe, place the agarose fragment in the spin column.

2. Centrifuge the tube at 2874 xg for no more than 45 seconds to elute the DNA. Spinning longer coelutes substances that are inhibitory to further enzymatic reactions. 2874 xg corresponds to 5500rpm for Eppendorf centrifuge model 5415; 5000rpm for model 5417.

3. Using a transilluminator, check eluent in bottom of outer tube for the presence of ethidium bromide stained DNA.

4. The eluted DNA can now be used directly in enzymatic reactions. For ligations, no more than 40% of the total reaction volume should be eluted DNA, as this reduces ligation efficiencies.

5. The DNA fragments can be further purified by ethanol precipitation. The addition of 2-4 micrograms of carrier tRNA prior to precipitation can increase yield, and its presence in ligation reactions is non-inhibitory.

or you may use the below protocol

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.

Best Regards

Sibtain

Weigh the agarose piece (1mg:1µL)

Add 1/100 of TE 100X and NaCL 5M to the final concentration of 200mM. (the total volumen shoud be 300 to 500 µL)

Melting the agarose at 65 Celsius degree by 20 min mixing by inmersion twice during de incubation

Add volumen/volumen of phenol saturated with TE1X

Vortex 5 min

Centrifuge 5min at 12 000 rpm

Collect the aqueous phase (upper) and concentrate it with 2 butanol (volumen/volumen) to 150-200µL

Precipitate the DNA with 1,2 volumen of isopropanol 88%/KAc 0.2M during 10 min at room temperature.

Centrifuge at 10 000 rpm during 5 min

Wash the pellet with ethanol 70%

Dry the pellet

Resuspend the pellet with 10-20 µL of water

Easy old-school way (though you may have to use LMT agarose and I don't know if TBE is a good idea). Cut out out the agarose strip containing the band of interest and put in small zip lock bag. Freeze at -20C. Squeeze the frozen bad through the bag with your fingers (gently) and remove the liquid with a micropipete. Clean up with phenol/choroform or EtOH precipitate if you have enough. Works for fragments of 1-4kb approx.

My question to sibtain afzal why high concentrated NaI is used? what's problem if we use low concentrated NaI?

Ive done the manual protocol as gustavo has mentioned, using freezeng the sliced gel with phenol, but i have gained 1microgram from 7 microgram run on gel. Is there any solution to enhance the efficiency?