PCR product looks less intense compared to others - should I increase the number of cycles? (Aug/05/2009 )
Hello all,
one of my PCR products has lower intensity compared to other PCR products with different primer sets. this product looks some times very faint. other primer sets work fine and give sharp bands.
Do I need to increase the number of cycles? I am running 35 cycles now. I also see a little smear above the bands, but not beneath it. the product size is ~1kB
35 cycles is quite high already and increasing the cycles might show you a stronger band for the faint band you are seeing right now, but it will definitely increase the smear for the sharp bands that you have right now.
Are you using the same conditions for all the PCRs (all of which have different primer sets)? Why don't you tweak the PCR conditions for the one giving you a faint band?
I doubt increasing cycles would help. I believe that the streaking could be due to your Mg2+ concentration. Try varying and see if that fixes your problem. Another factor could be your primer and template DNA concentration...
Curtis on Aug 5 2009, 02:47 PM said:
one of my PCR products has lower intensity compared to other PCR products with different primer sets. this product looks some times very faint. other primer sets work fine and give sharp bands.
Do I need to increase the number of cycles? I am running 35 cycles now. I also see a little smear above the bands, but not beneath it. the product size is ~1kB
In my opinion, 35 cycles is quite enough, increasing the cycles only lead to bad smear……
For one of your products has lower intensity, I think the problem maybe the template DNA, you can do a dilution of your template, or decrease the annealling temperature in your PCR process.
I assume you use same annealing temperature for all your PCR reaction. Try do a gradient PCR in the range of + and - of 5C of your current annealing temperature to see whether this is the sharpest band you can get. You might get a sharper band in higher or lower temperature. Also, try increase your annealing time 20 seconds more, and your externsion time to 1min 10 seconds. 35cycles is more than enough. usually 30 cycles will give you a distinct band.
The little smear above your band most probably due to too fast electrophoresis. I didn't see your gel and not sure your condition so I can't say much.
Hope this helps. 