I did a PCR assay and the PCR product is purified by magnetic bead purification. By running the PCR product on TAE agarose gel, I got the clear desired amplicon. But when I tried the purified product on the gel, I see nothing, however I used 50ng of DNA in the gel's cell.
I can not figure out why and what to do.
Zx Chong Hi, thanks for your reply.
I used QFX Fluorometer to measure the dsDNA concentration. And I used 50ng in each cell on the gel to make sure it can be visualized.
Maybe I need to use more. I don't know the real maximum and minimum amount of DNA we need for gel visualization. I thought 50ng would be enough.
Hi
50ng is not much for visualization in the gel. You could use a method which is more sensitive like TapeStation or QIAxcel to check the products without losing much of your DNA
May be the purification was not successful. You should try again.
Hi!
There are some little columns for PCR clean-up if you want to try a method not involving magnetic beads.
https://www.omegabiotek.com/product/e-z-n-a-cycle-pure-kit-v-spin/
You can also run all your PCR product on gel (from a large PCR reaction, like 50 or 100 µl reactions), maybe in several wells to fit it all, and then cut your amplicon band(s) and extract it from the gel (the electrophoresis will help to get rid of different size DNA such as template and primers). In the past I was using this method using this kit:
https://www.omegabiotek.com/product/e-z-n-a-microelute-gel-extraction-kit/
and then using the PCR fragment in plasmid construction with success.
Good luck
The recovery rate in PCR purification is ~70%. So, should try gel electrophoresis with a large amount of purified product.
Purification is not proper and you have lost DNA during purification. You can increase the concentration of DNA and visualized it with gel electrophoresis.
Hi,
Supposedly you can see the band with the concentration of 50ng on the gel (It will appear fade on the gel of course). There is possibility that the concentration of the purified product is lesser than that. You may want to amplify more and pool them together. Then load them on the gel. Once you see a very bright band on the gel, proceed with the gel purification step. After purification, I usually check the DNA concentration by running 3ul of the product on the gel as I don't have any access to Nanodrop or other DNA quantification machine.
Other option is to precipitate your purified product to increase the DNA concentration. However, if your starting purified DNA is very low, there is possibility you will end up with no DNA at all after precipitating it.
Hope this helps!
Thank you so much for all your replies.
Actually I have to stay with magnetic bead purification :) I'm doing my thesis. but thanks anyway for the other suggested methods.
Elena Fujiwara
, Shahanoor Alam May I ask how the amplicon size is related with this issue? This is sth that I may not know.Thanks.
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