Dear All,
My target gene have another variant and it was expressed too in my gel. my target gene is 404 bp and the variant 344. how to longer separate it. so I can cut my target gene and purification by gel extraction. what the best concentration gel may I use? How long running? thanks very much for your attention
Dear Dewi,
You could use nested PCR to get only your target gene. If not, you could cast a high concentration agarose gel (~ 2%) and take real good care while excising your band of interest!
Hi,
Theoretically, u shd be able to easily separate it using a 2% gel. Make sure you run it as slow as possible.
Another trick wld be running the gel till the band u dont need runs off the gel and u r left with the desired band.
I wld also consider using smaller sample load or optimizing the running buffer in case the aforementioned tricks will not work for u!
Check the following pages in case u need further info:
https://agctsequencing.wordpress.com/2012/02/14/dna-fragments-resolve-better-on-correct-percent-agarose-gel/
https://dk.promega.com/resources/pubhub/enotes/what-percentage-agarose-is-needed-to-sufficiently-resolve-my-dna-sample/
If these are PCR products and the rest of the gel is clean, you can very easily use a PCR cloning vector to directly clone both of the products directly from your PCR reaction and screen clones by digest/sequencing. Since they are similar size you should get a similar number of clones for each product, unless one band is more dominant than the other. Even if you co-purify both in a gel purification, and go straight into your final vector, the same logic applies. Just clone both and screen by sequencing.
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