Low concentration after gel purification - PCR product purification (Jun/10/2007 )
I use Qiagen kit to purifu the PCR product in the gel but always get low concentration after purified( around 2-4 ng/ul). If I purify directly from PCR reaction, I will get a very high concentration ( around 100ng/ul). I got this experience
ANybody has counter these kind problem? Help!
( I have to run gel for another PCR because I can't get ride of extra band by optimise the condition of PCR......)
Yes, if I purify from directly from PCR product the total amount of DNA is higher when compared to DNA amount obtained from isolation from gel but that is quite normal as you usually loose something like 20% DNA by runing on gel.
Although I don't get so low concentrations like you said you get.
Starting from a 25ul F.V. PCR I get something like 15ng /ul eluting with 15ul
In how much volume do you elute your DNA , maybe you can decrease the volume.
by the way why do you need to run PCR product in gel?
For cloning you can purify from PCR and digest it.
Is it because your PCR product is not specific and you need to cut the rigtht size band from the gel?
This can happen if the PCR product (starting product) is not a lot so the end product after purification is low.
may be this concentration is sufficient for ligation. Then it shouldnt be a problem.
I also experience the same thing.
Although I got low concentration reading from Nanodrop after gel extraction, when I run a little volume of the gel purified product on gel, most of the time, I got quite high concentration of my product. My hypothesis is, there could be some trace amount of EtBr left in the gel purified product that interferes with the Nanodrop from giving the accurate reading. Therefore, I seldom trust the reading from Nanodrop for the gel extraction product. I rather run the product on gel and estimate it with my naked eyes.
What do you think about it?
I used the Roche Kit to extract the DNA from the gel and now I have a lot of EtOH inside my samples. Do you know how to get rid of it?
I use one kit from invitrogen (PureLink, Quick Gel Extraction Kit) and I´m very happy with it.
And one very cool product is the Micropure-EZ from millipore. In one step you can ged rid of many different enzymes (restriction enzymes, polymerases, ligases...). have a look on the page: http://www.millipore.com/catalogue.nsf/docs/C7485.
I think this could be useful!!
Strangly enough by ethanol percipitating your DNA sample, followed by a 70% Etanol wash and spin. Once the ethanol is removed the DNA is left on the bench to dry. Once the EtOH has evaporated, then resuspend the DNA in TE or dswater.