I am trying to devise a quick method to extract genomic DNA from human cells for a PCR.
I first collected cells by centrifuging saline mouthwash (0.9% NaCl) and extracted genomic DNA using kit followed by a PCR protocol which works fine but is a rather lengthy process.
I tried to cut short the method and removed the DNA extraction steps by using pelleted spit samples as templates for PCR and increased the initial denaturing step from 3 minutes to 5 minutes. However this method was not reliable as some of my samples were amplified and some were not.
Can anyone suggest me a reliable protocol here?
If you have too much template it may inhibit the pcr. You can try pellet the cells, wash with pbs 1x, boil 5 min more or less and use this as template for pcr. Before using in the sample make a test with your own sample and other person that can contribute with sample.
The most common problem is having too much template (with inhibitors). Less is more. Dilute and use small amounts.