PCR product appear two close band in my gel! - (Jan/13/2009 )
I am newbird over here and currently working on bacterial DNA work. My research is based on extracting bacterial DNA inside zooplankton body and identified the species of bacterial that exist in the that zooplankton. I am using Boiling method for doing my DNA extraction work and used that Bacterial and Zooplankton DNA mixture to do PCR. However, 1 of my primer have give me extra band. The exact band size i want is around 500bp...but i am getting another band which is 20-30bp less than the band size that i should get. I have 2 band in my PCR product!!!I don't know what reason that cause this and anyone here can explain to me? Please!
are you sure your primers are specific? have you checked them against the genome?
you can try increasing the annealing temperature a little to prevent non-specific priming (you can try a gradient if you have a gradient cycler).
you can try increasing the annealing temperature a little to prevent non-specific priming (you can try a gradient if you have a gradient cycler).
Yes...my primer are specific ..i have try before ..from 55-58 degree..but the band still there..i also have change my MgCl concentration...however, i don't get any pcr product if i reduce MgCl concentration..
Left to Right
Lane 2,3,5,6--> bacterial+ zooplankton( different condition) 2 bands
Lane 4- pure bacterial----> 1 band