The DNA was extracted from spittle (saliva). The samples have very low concentrations (2-5ng/ul). I used DNA from blood as control positive but with the same concentration of the samples, and I get amplification only the controls, suggesting that maybe the samples have impurities. Does anyone has some protocol to amplify samples in these conditions?
Also would be good to check of any inhibitiors presence (i.e. polyphenols and some others). You also can try to apply different thechniques for concentration increasing. The simpest way is to precipitate DNA with alcohol and dissovle it in smaller volume of buffer or nuclease free water.
If you have problems with inhibitors usually diluting the sample and/or additional washing steps help, but if you already have low concentrations this can lead to new problems. Therefore perhaps additives such as BSA can help to overcome inhibitor problems. Betaine and the already mentioned DMSO may help for "difficult" samples as "reaction enhancer".
You had plenty of DNA if your concentrations are correct. I would consider dilution of the samples - if there inhibitors present, they will become diluted and have less of an effect
I would also go for dilution and a higher number of PCR cycles. We use the Taq polymerase from Bioline MyTaq HS Red Mix for environmental samples. There are also often problems with inhibitors but this Taq mastermix works quite well.
You can work with variants of taq. Unless you are amplifying a GC rich region, or a region with a lot of folds, I do not see a reason as to why you can't work with normal taq.
Try making the reaction comfortable. A couple of things that I would try are: work with a 5ng template in a 10-15ul reaction, work with varying concentrations of mgcl2, add a bit of extra primers, polymerase, etc so that the reaction is more comfortable. Also, use a lower tm. Once you have obtained bands, you can then use the pcr product to reamplify your target region using slightly stringent conditions.
Include 5-10%DMSO into your reaction. Sounds simple but may help.
Concerning polymerases, Phusion is worth of trying, but please use GC-rich buffer system.
People, thanks very much for your answers. Helped me a lot! In this week I going to try a new protocol, maybe I have to buy a taq as told by some colleagues. I will tell you the new results! Hugs!!!
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