Dna purification from gel - (May/27/2008 )
Hi, my name is Jose and I am doing a PHD. I am trying to extract DNA from gel using a kit(quiagene) but I am having problems. I do not obtain enough DNA, always < 15 ng/ul. The protocol is really easy and everyone tell me that they did not have any problem, but or I am stupid or I am doing something wrong.
I would like that someone give some advise. I would like to know if I can set up a big dye reaction without running my PCR product. Also, if I have to be carefull on any step during DNA gel purification. PLease I really need your help.
Thank you 
I would like that someone give some advise. I would like to know if I can set up a big dye reaction without running my PCR product. Also, if I have to be carefull on any step during DNA gel purification. PLease I really need your help.
Thank you
1. If the DNA fragment is less than 500bp, use isopropanol.
2. Whatever source of DNA you have, plasmid, RE fragment, PCR product, make sure you have run ample amount on gel to get enough after gel elution. Ie. If you run 750 ng, you can not expect more than 15 ng/ul after elution. If you have run 10microg, you can expect 100-200ng/ul, which should be good for sequencing.
3. Don't do any downstream application unless you are sure of DNA conc.
4. You can not use PCR product directly for sequencing, you need to purify it. If you have a very specific and strong amplification, you can do direct PCR purification (rather than running on gel), and submit a little for sequencing.
Well, Quiagen gel extraction kit is not 100% efficient. I usually get 50% recovery. It also depends on the size of your fragment.
You could try to start with more material.
If you are going to do ligation with your fragment, 15ng/ul might be enough.
I would like that someone give some advise. I would like to know if I can set up a big dye reaction without running my PCR product. Also, if I have to be carefull on any step during DNA gel purification. PLease I really need your help.
Thank you
1. If the DNA fragment is less than 500bp, use isopropanol.
2. Whatever source of DNA you have, plasmid, RE fragment, PCR product, make sure you have run ample amount on gel to get enough after gel elution. Ie. If you run 750 ng, you can not expect more than 15 ng/ul after elution. If you have run 10microg, you can expect 100-200ng/ul, which should be good for sequencing.
3. Don't do any downstream application unless you are sure of DNA conc.
4. You can not use PCR product directly for sequencing, you need to purify it. If you have a very specific and strong amplification, you can do direct PCR purification (rather than running on gel), and submit a little for sequencing.
Hi, thank you very much for your answer. My DNA is 900 to 1000 bp and I got a clear band, I do not see any contamination, unspecific sequences or primers. I will try to make the purification of my PCR product.
How can I know the amount of DNA I am running in the gel??, Do I havo to take a meassure before the electrophoresis??.
Finally, if I have a clear band, after purification do I need big dye reaction or I can send the PCR product purified directely for sequencing??
Thank you very much, you were very helpfull.
You could try to start with more material.
If you are going to do ligation with your fragment, 15ng/ul might be enough.
Hi Zek, thank you for your answer. I need my product for sequencing and my DNA is 900 bp long. MOre material mesn that I have to use different columns because I can not use more that 400 mgr gel per column. May be I can use more than one column and later join the product??? Could it be good idea centrifugate the sample several times once I added the water or the elution buffer??
Thank you
For sequencing I'll recommend the PCR purification kit. I had used the gel purification kit and the yield is very low and sometimes had trouble with the sequencing. But if can't change kit use a little bit more of alcohol sometimes the solution is to viscous?? and the column clogs and for the the elution step warm the buffer and incubate for 3-5 min (I usually add 1/2 incubate, centrifuge then repeat).
Hi, thank you very much for yor answer. Could you tell me the incubation temperature??
Thank you
It might be worth running 1 or 2 ul of the PCR product (before purification) to see if the problem is in the PCR reaction or if you are loosing tons of product in the gel purification. If the band is weak before purification, you may need to set up larger PCR reactions or optimize the PCR to make a more robust reaction. When you cut the pcr product from the gel try to cut off as much agarose that isn't stained with EtBr. For example, the small amount of gel under the wells will never have DNA and just adds to your limit of agarose. If you cut this off, you can include more gel with DNA in one column. Run the melted agarose/QG buffer through the column at least twice (after you spin 750ul through the column, pipette it up and run it back through the column again). Make absolutely certain that there is no remaining PE wash buffer after the second spin. This will inhibit elution. I spin mine for about 3-4 mins to make certain the filter is dry. Finally, elute in a smaller volume (about 30ul) of warmed water/elution buffer.
Depending on what you are using the PCR product for, 15ng/ul might be fine. For a ligation, this concentration is desirable. You can sequence your purified PCR product but don't forget that you don't get reliable results for at least 20 bases so you won't be able to check the sequence at the end that has the primer (unless you do both a forward and reverse). Otherwise, you may need to pool products from multiple columns and EtOH precipitate. I would consider this a last resort, however. When you mix the products from PCR reactions (multiple tubes) you run the risk of mixing a mutated PCR product with a good one.
I may be wrong here (please let me know if so) but the PCR purification kit will not remove the template DNA put into the PCR reaction. Can you digest a PCR reaction with DpnI to cut up the template and still get a pure PCR product or will there still be fragments from the template? Seems like this is iffy and may result in major problems in down-stream applications.
1> I was told by the local QIAGEN rep to heat the elution buffer to 65C. This has certainly increased the yields to~50% Still not great, but better than the ~305 we used to get.
Alternatives include the Geneclean kit which uses silica beads. Typical yields for my lab are 90%.
2. I agree with the idea of DpnI digestion to get rid of template. There may well be fragments, but as the termini are different to your insert, they shouldn't be a problem. This can also be helped by using less template.