to extract DNA from agarose gel - (May/30/2006 )

I have heard about the way to get a DNA fraction out off the agarose gel by placing the membrane in the gel in front of the DNA band after doing the electrophoresis for awhile and then doing an additional electrophoresis to drive the DNA onto the membrane....

So I have some questions:
What knid of membrane do they use on this kind of work?
Could I use a nitrocellulose membrane?
Could they elude the DNA out of the membrane later? and How?

Thank you so much
L Lim

---

why not just do glassmilk extraction, much quicker and very reliable

---

Dear Veteran
I'm quite new in the field...also quite low in budget! and I've seen nitrocellulose and nylon membrane using for colony hybridization sitting around the shelf with nobody using so I thought I could make use out of those stuff nobody wanted. Do you know the way to make use of the membrane to get the DNA out of the gel?
and by the way, what is the glassmilk? would you please give me more details/protocols on how to use it?
and If anyone have any idea please let me know... Thank you

L Lim

---

Hello LLL1969,

I can't say anything about the membrane technique, having never done it, but I can suggest a very "quick and dirty" method for extracting DNA from agarose gels. I don't know what you want to do with the DNA, so this method may not be good for you.

1. Cut out your band from the gel and trim off any excess agarose.
2. Place the gel slice in the top of a 100 ul filter pipette tip (some filters prevent any moisture passing through them, so don't use this type - most filter tips should be OK).
3. Cut off the bottom of the tip so it fits into a 1.5 ml Eppendorf tube (and doesn't collapse during Step 4).
4. Put the tip into the tube and centrifuge at 14,000 rpm for 30 seconds.
5. Remove the tip and voila - your DNA is in the gel buffer at the bottom of the tube. The agarose is blocked by the filter and stays at the top.

Remember that your DNA will now be in whatever buffer you make your gels with. Yield is fairly good, but I've never done this with DNA fragments larger than 1.5 kb. The whole process takes about 5 minutes and is cheap (1 tip, 1 tube, 1 scalpel blade).

Hope this helps.

---

otherwise you can check the protocol online site

http://www.protocol-online.org/cgi-bin/pro...rose+extraction

here is a list of extraction procedures, glassmilk extraction is usually done with a DNA extraction kit, possibly more expensive but check the link for some other ways

also note that as said above the purity of DNA needed depends largely on what you plan to do with it afterwards. So for things such as cloning etc, the DNA needs to be pretty pure.

also useful is to use less EtBr than normal as this can hinder further DNA manipulation, so you can always stain a control lane and use that to judge the area of agorose where your DNA is present

---

Thank you wbla3335 and grapes
After all whining... I did check out the protocol pages of this web site also the the link Grapes gave me...
And I found this

But I have no access to any of these paper
Would anyone check these protocol for me please? I would prefer Hansen's method the most.
thank you everyone inadvance...and
Thank you wbla3335 and Grapes of wrath very much

Oh...by the way... my work is involving the cloning of somewhat big DNA fragment extracted from soil-clay (10 kb up, favored bigger than 40 kb...but with my lacking skill I can't extract that big of DNA yet )

---

Hi

If your budget is low, there's a method that I've tried before and it's very simple.

1. Cut agarose gel slice containing your PCR product (or DNA fragment). Transfer the slice to a 1.5 ml tube. Add 50 ul TE buffer into it.
2. Freeze in liquid N2 (or at -80C, >1 hr).
3. Spin at max speed for 10 to 20 min. Transfer supernatant to new 1.5 ml tube.
4. Add another 50ul TE buffer to the gel pellet. Resuspend the agarose gel. Repeat freezing step, and spin at max. speed for another 10 - 20 min. Pool the supernatant in 1.5 ml tube. [Normally, I'd just repeat the freezing and squeezing (centrifugation) once. You can repeat a few times to get more DNA].
5. Precipitate the pooled supernatant containing DNA using standard protocol (Sambrook et al). Note: Some suggested adding glycogen as DNA carrier, but I don't.

CAVEATS:
1. Make sure you're not using smaller tubes, i.e. 0.5 ml tube. They can't stand the pressure and will crack, leaking the content out during centrifugation. That's why I use a sturdy 1.5 ml tube.

I've used the purified DNA from above method to do sequencing and cloning without problem. That's when my budget was tight. Do find the following references useful, k:

1. Thuring et al 1975. A freeze-squeeze method for recovering long DNA from agarose gels. Analytical Biochemistry. 66(1): 213-220

Diethard & Renz 1983. An optimized freeze-squeeze method for recovery of DNA fragments from agarose gels. Analytical Biochemistry. 132(1): 14-19

Link from Northern Illinois Uni., Dept. Biological Sciences
http://www.bios.niu.edu/core/freeze_squeeze.htm

---

Thank Isek
I'll check them out and will give it a try.

L Lim

---

A good technique,I have done this according to the access,but inefficient!

---

Try out following protocol, works fine for me:
ELUTION OF DNA FROM AGAROSE GEL ( FREEZE-THAW METHOD)
1. Cut gel with the desired band on a low intensity UV with sterile sharp.

2. Put the cut gel in an epp tube and freeze at –70o C for 15 min.(Max 24Hrs)

3. Melt gel on Heat Block (5 min,65C) & add eq vol TE sat Phenol. Gently crush gel with tip.

4. Vortex gently and freeze at -70°C for 15 min.

5. Thaw the tube and spin at RT for 5 min at 12000 rpm.

6. Take out the aqueous phase in another clean tube and add equal volume of Chloroform: Iso Amyl Alcohol, (24:1). Spin at RT, 12,000 rpm, 5 min.

7. Take out the aq phase carefully & add 3M Sodium Acetate (pH-5.2) so that the final conc of Sod Act is 0.3M.

8. Add twice the vol of prechilled abs ethanol. Mix well by inverting. Inc at -70°C for 30 min. (Max24 Hrs)

9. Spin @ 13,000rpm, for 10min at 4°C. Remove SN and wash the pellet with 70% pre chilled Ethanol(500μl). Mix well, inc at RT for 5-10 min.

10. Spin @ 13,000 rpm, 7 min at 4°C. Remove the SN, Air dry the pellet at RT and re suspend in 20/40μl of TE/MQ.
Or
if you want to use membrane check the protocol in the picture attached( I used Sigma dialysis tubing (cellulose) cat# D9777

---