Currently I am trying to troubleshoot difficult extractions. These extractions came from a wing punch from a bat. I am struggling to amplify the DNA through PCR. I have reduced the amount of hydrase these extractions had in order to increase concentration of DNA. This allowed a Qubit to detect the presence of DNA. The concentrations are anywhere from 1 to 3 micrograms per milliliter. It also appears some sort of inhibitor is present. Any methods on amplifying low concentrations of DNA with inhibitors would be great.
It may sound counterintuitive, but sometimes diluting out the DNA even further will help. PCR is very sensitive and the enzyme will be able to amplify the DNA once the level of inhibitor is low enough. You can also try adding DMSO to your PCR mix or buy an enzyme mix that uses DMSO.
You just prefer Mega Yield DNA kit. You can isolate the large template of DNA from small quantity of blood samples.
you can try using a speedvac to concentrate your DNA further, however this will also concentrate any other unwanted substances (inhibitors) that is in your DNA solution. Also you could try ethanol precipitation using salts to further concentrate your DNA, this will get rid of some of the unwanted substances in your solution, but you do run the risk of losing some of the total DNA amount.
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