If PCR generates a smear after running the products on a gel, what can be done to improve the results?
If PCR generates a smear after running the products on a gel, what can be done to improve the results?
First, determine the source of the smear using positive and negative (no template) controls. This can determine if the cause of the smear is contamination or overcycling, or if the smear results from poorly designed primers or suboptimal PCR conditions.
If the negative control is blank, there is no contamination. Instead, the PCR conditions will need to be optimized; consider the following when adjusting the PCR conditions:
-Reduce the amount of template.
-Increase the annealing temperature.
-Use touchdown PCR.
-Reduce the number of PCR cycles.
-Redesign the primers.
-Use nested primers.
-Re-amplify the product. (A small plug of the gel can be removed with a micropipette tip, and the DNA can be recovered by adding the plug to 200 µl of water and then incubating at 37°C. 5 µl of this solution can be used as PCR template for re-amplification.)
If the negative control is also smeared, there is contamination. You will need to determine the source of this contamination. It may be necessary to replace PCR reagents and to decontaminate pipettes and your workstation (see questions below for more information on contamination).