@Enrique: That usually depends on the number of colonies you want to screen. When you do a simple colony screening, you can easily test hundrets of colonies. Doing a DNA extraction on all of them first costs a lot of time and is also a bit painful.

Instead of dipping the colony directly into the reaction mix you can dip it in 20-30ul of water,boil it for 10 mins, centrifuge down and use the superantant containing dna(5-10ul) for PCR. this removes the unwanted inhibitors and enhances the PCR quality.

Well fungi may produce inhibitors of polymerase in the growth media in the form of secondary metabolites. You need to think about removing these. And I trust you use an internal amplification control for your PCR. (I´ve published on these.)

Note ammonium sulfate is acidic and KCl is netral and that made the difference.

Add KCl it might be important for fungi cell wall lysis

Thank you all for your answers, especially Ted and Jai for giving me hints on the differences between those substances.

It turned out that my student colleague who was carrying out the work, forgot to add BSA (1% final conc.) to the reaction (the previous recipe with KCl included that) and now that we found out, it´s working again.

Almost embarrasing, that I didn´t check before. Sorry, but at least I now know about the Hofmeister series, which I hadn´t heard before. :-)

I can suggest re-suspending about 1/10 of a 1mm colony in 25 uL of freshly diluted (from 10M stock) 0.03M NaOH. Boil this suspension for 5 to 10 min and place on ice.

Prepare your PCR mix as usual (25 uL of reaction is enough for a diagnostic PCR), with standard Mg concentration. Heat the PCR block to 94oC (hold), place the empty PCR tubes on the block, add 1 uL of the cell suspension to each tube, add the 25 uL of PCR mix to each tube, close the tubes and re-start the program. It works well even with old colonies in YNB, YPD, or synthetic medium.

I agree the sugestion of elizabeth bilsland

Thank you for your suggestions, but as I already mentioned: The key was simply to add 1 % BSA into the pcr mix. No other pretreatment necessary (and we do want to save any time we can).

@ Anna Albers: Thank you, but the method we use is intended to circumvent DNA extraction and go directly from culture to PCR-product. Saves us time and money.

Dear Dresch,

for fungal DNA extraction, I think you can use the following paper " Leach etal.1986. Rapid mini-prep of the DNA from filamentous fungi. Neurospora . Newsletter. 33:32-33."

for that, I think some the problems that you faced can solved by changing the solution that used such using LETS buffer for fungal cells lysing and DNA extraction which contain 0.1 M LiCl, 10 mM EDTA, 10 mM Tris-HCl pH 8.0, 0.5% SDS.

Best