In general, purified DNA should serve as a good template for PCR assuming primers are complementary with the target especially in their 3' sequences. What organ are you extracting DNA from? Do you get good DNA purity, say, good A260/280 ratios, yields? Assuming there's no problem with template purity, trying a nested PCR approach can help by designing out-flanking primers in the same region. It's also possible that the target region might be GC-rich and thus it zippers back easily making amplification difficult. Trying a biphasic PCR with or without betain could help by merging the annealing and elongation steps but it's important to have primers with higher Tm. 

Hi Oscar,

I have extracted the DNA from the tail and recently from the head of the tadpoles. There are not very big so I did what I could do. My DNA 280/260 ratios are between 1.8 and 2. I have used BSA and with this I achieved to amplified and get one single band in Rana arvalis. Also, I have tried DMSO 5%, I am gonna try now a gradient till 10%. However, R. temporaria is resisting...Do you know the concentration of betain that I should used for this?

Hi Maria, thanks for your reply, your DNA ratios are excelent, the choice of DMSO is a good one, I'm not sure if betain can improve whatever results you get but here's a methods paper (see link).   Trying different primers is important and very cheap to do. In PCR like other things in life, size matters and thus if the fragment you're trying to amplify is too big then getting a Taq polymerase with good processivity and leaving enough time for elongation is important (1 min / 1 kbp). I'm not sure about the choice of using BSA, it might contain a PCR inhibitor, don't know. I would leave it out if possible.

Oops, the link didn't work.

https://www.promega.com/resources/pubhub/promega-notes-1998/betaine-and-dmso-enhancing-agents-for-pcr/

Thank you very much for the link. I will take a look carefully! :).

Should I take care about the 260/230 ratio?. Today,  I have been measuring DNA concentration from my last DNA extractions. The   280/260 ratio is fine, however the 260/230 ratio is lower than 1.8 in most of the cases. In the tutorial says that it is a secondary measure of quality. How can affect this to my samples. and if it is lower, How can I get rid off  the impurities?

I would not worry  too much about a ~1.7 A260/280 ratio especially if you measured it in a different spectrophotometer from the one you got higher ratios before. But if you want to clean it up the easiest way is to EtOH-precipitate by first adding 0.1 volumes of 3 M Sodium Acetate to your DNA sample (to neutralize the DNA polycation), mix throroughly but gently to avoid shearing of DNA and then add 2.5 volumes of cold Absolute ethanol (to generate a hydrophobic environ). Mix gently and you'll see like a bugger that forms upon gentle shaking. If you don't see anything, place the mix at -20 degrees C overnight (just to be on the safe side) to allow precipitation to occur. Next, centrifuge 15 min at top speed (12,000 g) in the cold. You should see a pellet that you can clean with one or two washes of 75% ethanol. In the last centrifugation make sure to aspirate completely the ethanol using a pipet and leave the pellet to almost dry and resuspend in Tris EDTA.  - If the ratio does not improve it means that it has contaminants more related to the DNA isolation method and for most practical purposes it doesn't interfere with PCR.  Good luck.

Thank you very much. I will do it tomorrow morning :).

Hi Oscar, 

The cleaning with Na-ac it worked pretty well. At least to samples from Germany. I am extacting a bunch of different samples from different localities to see if the problem will be solved in this way. Today, I have started the Na-ac cleaning for these new samples and the centrifuge machine did not work properly. I did not cool down to 4 degrees, It cooled down until 10 degrees. Do you think the temperature it is very critical at this stage?

Thank you Very much! :)

Hi Maria, great news!. This protocol is the less intensive, if your samples have a lot of protein you could use another one relying on Proteinase K with later Phenol Chloroform extraction to get rid of the Proteinase K and original contaminants, however it requires a lot more reagents, more steps and with the inconvenience of handling Phenol which is not nice to have around. -  Regarding your question about the centrifuge temperature I don't think it matters. If you have a good chunk of DNA it should precipitate soon after adding the Ethanol. I believe that if the DNA has precipitated, further centrifugation at room temperature doesn't matter.

Thanks a lot!. Great then!. I will measure the DNA when ethanol is totally evaporated which is an extra problem in frogs. It takes two days until everything is gone! I have extracted different kind of animals but this ones are very tricky! I hope it works, it does not! I will try phenol chloroform extraction. Thank you again for your help!