I extract DNA from sweet potato and amplify it with v1-v3 16S primer then I re-amplify it again with GC-clamp to use it as samples for DGGE. The problem is, the result of GC-clamp primer PCR contains smears and too much primer dimers. So I purify the DNA with gel extraction purification KIT. However, right now I am running the sample in DGGE 80volt with 40-60% denaturant in 6% gel yet my samples don't turn down well. The loading dye turn down as smears in the gel. I wonder why and what I did wrong and what I shall do next.
Thank you!
Hello,
May I ask what are your PCR conditions? Do you have a picture? As you state you had primer dimers and PCR product smears, potentially your PCR conditions are inadequate thus affecting your downstream DGGE analysis. This may be caused by poor cycling/temperature and or poor PCR product. You can refer to these two websites for specifics.
http://www.bio-rad.com/en-us/applications-technologies/pcr-troubleshooting
http://ddgehelp.blogspot.dk/2005/11/dgge-guide.html
Hope this helps.
Juan
Quite basic error- why did you use bacterial primers for amplifying sweet potato DNA?? Try reading the procedure here:
http://www.scielo.br/pdf/sa/v66n4/a15v66n4.pdf
Hii..
If you are getting smear after PCR amplification then you need to optimize the annealing conditions.. Generally Primers for V1-V3 works well at 53-55 degree. Also one thing is that the annealing conditions will be different for the primers with and without GC clamps. For more help, gel with PCR product will be good..
Best wishes.
Try to check after amplified with v1-v3 16S primer,may be the concentration of primer more.
Reduce the concentration of GC Clamp which may be the reason.
Hi Juli,
As a control to check that your gel is OK you can run a standard DNA molecular weight marker. We have used 100bp GeneRuler from Thermo Fisher, but you could try 1kb as well. Use standard volumes as you would load an agarose gel. You should get distinct bands, although they will not separate in the same way as in agarose of course. If this results in smearing, it could be a problem with the gel.
We have seen problems with smearing when the gradient doesn't form properly.
We have also found that GC clamps aren't always necessary. Try running product with and without GC clamps. Primer dimers could be forming because the GC clamp may provide a lot of 'stickyness' for the 3' end of your opposite primer.
Check out http://ddgehelp.blogspot.dk/2005/11/dgge-guide.html to troubleshoot other problems.
Regards
Rich
Hi Juli,
it seems that the first and main problem comes from PCR amplifications, because if you get amplicons with a lot of pirmer-dimers and smear, as you describe, it is quite normal to get not a good DGGE run. This problem could be due to all the factors mentioned by the previous answers, in addition check well which primer to choose for attaching GC clamp.
In addition if you see a smear with simply running the loading dye, this I think may Happen because if the dye is not intercalated to nucleic acid it tends to spread in the gel.
Regards