First I think the size was the problem because Quiagen-Kit works only till about 10kb. Did you check ethanol? Because one of the solution have to put ethanol inside, it is not ready to use when you open a new kid. Another problem is sometime that in kits which are opened for a long time the ethanol concentration decrease.

What you also can test is to go down with the volume of the last step where you wash down the DNA from the column. For example use only 50 uL. And then first put 30 uL to the column, wait 2 (not 1 minute what is often written in the manuals of such kits) minutes and then centrifuge. After that put 20 uL to the column and wait again 2 minutes and centrifuge.

I use QIAEX II Gel extraction kit. It has some kind of beads instead of a column. I can recover more DNA than compaired to columns. And also I try to make digestions with small final volume, so you can load in only one slot of your gel. For instance, you can digest 2 ug of DNA with 2 uL of enzyme in a final volume of 30uL reaction and you load in one single slot of your gel, avoiding too much agarose. The limit is 250mg of agarose per reaction, so usualy this means for me two slots of my gel. 2 slots with 2ug of DNA each should be fine for having a good final DNA concentration to use after for a ligation for example.

I hope you are using GEL extraction kits, and not PCR purification kits, as you wrote :)

good luck!

You say you are trying to recover a fragment from a gel using a PCR purification kit? Qiagen has 2 different kits. Even thought the columns are the same, the gel extraction kit uses QG buffer to dissolve the gel, whereas the PCR purification kit uses PB or PBI buffer to dilute the PCR sample...

Also another tip: when you add ethanol to the buffer, don't use 200-proof ethanol, but rather regular ethanol 96% ethanol. For some reason I had bad recovery when using 200-proof ethanol.

For such a large fragment, I would use a low melting agar. You can always use the old good glass wool system. It's an old method and the recovery is not as good as with the kits for smaller size fragments, but it works as a charm for larger ones.

Recently a group from China published a variation on it.

Here is the link to it:

http://www.jcancer.org/v03p0093.htm

Good luck!

Hi! I recently had to purify total DNA from agarose gel. Due to DNA size, the commercial kits were not recommended. Thus I had to use an old-fashion protocol which revealed very efficient:

1/ melt the excised gel at 65°C for 10 minutes;

2/ add 0.5X aquaphenol;

3/ centrifugate at room temperature, 14,000g for 5 minutes;

4/ recover carefully the aqueous upper phase;

5/ repeat once steps 2, 3 and 4;

6/ bring the solution to 0.3M NaAcetate;

7/ add 0.7 vol Isopropanol;

8/ incubate at -20°c for 30 minutes;

9/ centrifugate at 4°C, 15,000g for 10 minutes;

10/ immediately and carefully discard the supernatant;

11/ add 1ml 70% frozen ethanol;

12/ vortex briefly;

13/ centrifugate at 4°C, 15,000g for 5 minutes;

14/ immediately and VERY carefully discard the supernant;

15/ let the tube open under a hood for 5 minutes to dry the DNA pellet;

16/ suspend in storage buffer or water.

I can answer questions if you need. Do not hesitate!

Good luck!

By the way, I agree with Ryan, Low Melting Point agarose helps a lot!

Use a gel extraction and not a PCR purification kit. Run the fragments on a low % agarose gel and check that everything is melted. Check also what is the max plasmid size and the max DNA quantity of your purification column. Good luck

The Qiagen kit for this works the best and is good for further cloning work too..!!

Hi everyone! Thanks so much for all the replies. Much appreciated. Yes, I used gel extraction kit (sorry for my mistake in posting). I checked very carefully every time that the gel is properly melted. Fragment size wouldn't be also a problem here, since 3KB plasmid dint work too. But yes, I think the ethanol concentration of the washing buffer could possibly be the problem. I will give solvent extraction (using low melting agarose) a try. Hope the quality is good enough for ligation afterward.

I always manage to extract using the Fermentas gel extraction kit. I think for that size you will need to add ethanol in the optional step. Hope this helps

Many good points already, I thought I would just add a couple of things that might be useful.

I use routinely both Qiagen and Macherey-Nagel gel extraction kits for fragments above 10kb with no problems. And so do many others in my lab. At some point I even compared column vs no-column kits (such as Qiagen EX II) and I did not observe any major difference.

For elution you can try two 25-30uL elutions with 3-5 min incubation at 60C, that works very well for me when I need high recovery.

Also, consider that you always lose a lot of DNA when purifying from gel. How much did you load to begin with?

Good luck

As a side note, a lot companies now make PCR/Gel extraction kits, that can be used for both applications.

Yes, I also suggest you incubate at 60C 2-3 min after you add water. other thing is you could reuse this water for next tube then you will concentrate it.

Try another agarose ( a new box), use fresh autoclaved H2O.

I've always had good success with the gel extraction kits, but there are other methods which might work better for large fragments. One is using beta-agarase, an agarose-digesting enzyme. It's available from NEB, USB, and probably others. If you want to be extra-careful, you could use the beta-agarase to release your DNA from the gel, then use the gel extraction kit to clean it up.

The other technique is to cut a well into the gel in front of your band, then run the DNA fragment into the well. You can use a piece of low-molecular-weight-cutoff dialysis tubing at the far side of the well to prevent further migration of the DNA. Then you briefly reverse the current to remove any DNA stuck to the tubing and remove the DNA-containing buffer in the well. Note that you should lower the level of buffer in your gel apparatus before doing this.

This and other techniques are discussed in a classic TIBS article archived here:

http://rothlab.ucdavis.edu/protocols/DNA-from-Agarose-Review.html

Please feel free to try Biomiga's Gel Extraction Kit, offered by GeniCity.com, you can ask for a sample kit to test, email [email protected]