I did PCR for seven MLST genes, but on electrophoresis the ladders bands didn't follow the length of my genes bands.......for example: my target band length is 500bp according on literatures but the ladder says it's 800bp!!!and did so with the other six genes (significant discrepancies between the ladder and genes). On the other hand, the same ladder works well with other genes and gives the exact length.
I repeated the electrophoresis again using different ladders against my MLST genes but same problem......Then I sequenced the gene and confirmed the length as 500 .... and sequenced all genes and confirmed their lengths.
So, is there any suggested explanation?
Thanks
One possibility would be that your ladder has a way different salt concentration than your PCR mix: Salt can affect the migration pattern: http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_BenchGuides_SourceBook_Section_III_-_Loading_and_Running_DNA_in_Agarose_Gels.pdf
Did you sequence the gene or the PCR product? Could it be that you have a repetitive sequence and hence the PCR products fuse during amplification? Eg the newly made PCR product can also bind on itself and serve as primer?
One possibility would be that your ladder has a way different salt concentration than your PCR mix: Salt can affect the migration pattern: http://bio.lonza.com/uploads/tx_mwaxmarketingmaterial/Lonza_BenchGuides_SourceBook_Section_III_-_Loading_and_Running_DNA_in_Agarose_Gels.pdf
Did you sequence the gene or the PCR product? Could it be that you have a repetitive sequence and hence the PCR products fuse during amplification? Eg the newly made PCR product can also bind on itself and serve as primer?
Change in buffer's ph and concentration can affect DNA mobility. Please check these parameters.
Check the ph and concentration of your buffer solution and use a fresh solution each time.
the difference can occur if you supercoiled DNA and linear DNA for example if you run plasmids and a linear ladder. best wishes
- Badges
- Science method