I have just performed Direct PCR on some of my cyanobacterial cultures and would like to send the PCR product for sanger sequencing. I ran a gel and got very smeared bands. Do you think these will be ok when I send them out for sequencing? Any advice on increasing the quality of my direct PCR product?
Your gel looks like the agarose has not dissolved properly. Heat the agarose for longer and swirl the gel to make sure that it is setting smooth without particles in it. Re run a sample of the pcr product in this new gel or on a colleagues gel and if the pcr band is tight and clean then it will sequence well
I recommend treating the amplicon prior to sequencing to remove unincorporated dNTPs and other artifacts of the PCR. When we generate PCR products in our lab, we treat them with a product called Exosapit from Thermo-fisher. There are also commercial kits that can purify bands that are cut from low-melting point agarose. I'm not sure it would be a good idea to send the raw amplicons for sequencing without some firm of cleanup.
Joel D Anderson Thank you so much! I will look into PCR clean-up kits.
Hey,
You cannot send direct PCR product to be sequenced due to the fact that it contains non binding primers as well as dNTPs.
You must isolate the expected band by cutting from agarose and purifying. direct PCR purification might cauuse a problem as well if you have primer dimers or non spesific amplication.
You can use M&N gel extraction kit it is cheap and quite well.
- Badges
- Science method