DNA Extraction from gel.

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johnsmith
johnsmith's picture
DNA Extraction from gel.

Good day everyone. I have a question which I hope someone could help me solve. I did a PCR, it worked great. I can see the bands on the argarose that is no problem and the amount of DNA is also what I would like to have. My problem lies with the extraction from the gel. I simply do not get the amount of DNA I would like that is if I could believe the Photometer. If I do a ligation my plasmid also seems not to work as my cells do not grow. This gives me a suspicion that the problem lies with the gel extraction.

I am currently in China and the kit I am using is from BioDev it is the B Type: Mini-DNA Rapid Purification Kit. Does anyone has previous experience with this kit or could tell me what I could be doing wrong?

Any help or suggestions would be appreciated.
John

itaylor
itaylor's picture
I'm not familiar with that

I'm not familiar with that brand, but I assume it works like other kits used for gel extractions. One very important key is to excise your band with as little agarose as possible. Then make sure its fully melted before continuting your extraction. Finally ensure that your Isopropanol/EtOH (whichever your kit calls for) is not old. Gel extraction kits are not very efficient to begin with so, try starting with more product before beginning any extraction (2-3 rxns worth).

johnsmith
johnsmith's picture
Thanx for the advice. Ill

Thanx for the advice. Ill keep trying!

cmartino
cmartino's picture
When you visualized your gel,

When you visualized your gel, did you use low UV? If not, you may have damaged the DNA with a high UV transilluminator before cutting out the band.

magdalena
magdalena's picture
johnsmith wrote:

johnsmith wrote:

Good day everyone. I have a question which I hope someone could help me solve. I did a PCR, it worked great. I can see the bands on the argarose that is no problem and the amount of DNA is also what I would like to have. My problem lies with the extraction from the gel. I simply do not get the amount of DNA I would like that is if I could believe the Photometer. If I do a ligation my plasmid also seems not to work as my cells do not grow. This gives me a suspicion that the problem lies with the gel extraction.

I am currently in China and the kit I am using is from BioDev it is the B Type: Mini-DNA Rapid Purification Kit. Does anyone has previous experience with this kit or could tell me what I could be doing wrong?

Any help or suggestions would be appreciated.
John

Hi.
I don't have experience with that particular kit, but I have had same problems with other rapid purification kits. It usually works if you incubate every step for longer time than indicated in the protocol. I usually incubate 30-40 min in every step.

labrat
labrat's picture
If you can get hold of it,

If you can get hold of it, try Qiagen's gel extraction kit.

I've never had it fail.

Also - have u tried running a small quantity of your extract back on a gel? Maybe try acrylamide and silver staining? That way you can see if there is DNA there and it's just your ligations that aren't working.