CTAB of Fungi - (Jul/11/2012 )

Sometimes with these sorts of reactions, especially with plants and fungi, they contain a lot of inhibitors that may cause the reaction to not work if the DNA prep is used directly in the PCR, however they may well work if the DNA prep is diluted, often 1:500-1:1000 seems to be a good range.

I did my PCR and ended up with a single bright band (even in NTC), very small - near the level of the last band of my 100bp ladder. Am I looking at contamination or primer dimers? Absolutely nothing else was amplified and there are no smears. Just this one band in every lane. If so then I think I'll have to convince the two I'm working with the primers are no good and we need to try something else. I did everything in a flow hood I cleaned with a DNexitus plus solution, pipette tips straight from the autoclave too, sadly my pipettes are not autoclavable.

I suspect you have primer-dimers. I never autoclave tubes. In my opinion, dirty autoclaves are the last place you want to put something that is supposed to be DNA free. Out of the box, they are essentially DNA free, especially if you are not working with human DNA.

This is awfully strange, do primer dimers often mean no desired amplification? I would have thought I'd get them with some amplification. I did the same thing the previous PCR attempt and got desired amplicons and a bit of smearing so this time I was using different template amounts of 20ng 50ng and 100ng to compare them. The only thing I think I did differently was I added my primers to the buffer and dNTPs before template (on ice). The last time I dotted my primers and taq onto the side of the tube (I still did it with taq) so as upon heating they fall into the buffer, template & dNTPs and start the reaction. People have described making master mixes with primers in though so I didn't think it mattered much.