Hello guys,
I need your help. I used to do PCR for genotyping of my LCK Cre mice. The PCR was working well. You can see in Fig A (Attached picture). No template NT was clean and negative control was negative. The positive band was 300bp. But as you see in Fig B my NT has 2 bands which one of which is similar to the expected positive band! I cleaned all the equipment and changed the material and repeated the PCR but still, there are bands in NT and negative control Fig c.
I really appreciate your help to figure out the problem.
Best regards
When your NTC and - samples show amplification bands you have pcr contamination in a reagent or in the pipettes or equipment that you use. Amplification of even tiny amounts of post pcr product is very efficient and you get over amplification which can over amplify both the correct band and very minor false bands and make them look strong. Often the amplimer becomes a smear. You cannot trust your results. Use all new reagents,plasticware and equipment and run the pcr in a different area of the lab away from where you usually work. If you design one or both primers to amplify a larger amplimer than your current one the problem will disappear and you will get good results again until you get contamination with the new primers
Respected Sir. Some times it happens, the unintentionally loading of PCR product in negative control. The PCR picture is showing clear band in negative control. It needs to run the gel again, as for as extra band may be dimer. And the upper band is the expected amplified PCR band while lowers bands are of the dimers.
41 cycles is a lot. Almost anything will amplify given that many cycles.
Have you tried this with 30-35 cycles? If you aren't seeing your desired band after 35 cycles, you should probably consider re-designing your primer set. Doing so may improve your signal to noise ratio.
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