Hi Said, personnaly, I performed colony-PCR on Rhodococcus and Gordonia species, which belong to the same sub-order of Corynebacterineae (Gram +, high GC-content, V-shape) as follow :

- prepare a pre-mix containing AmpliTaq Gold 360 MasterMix, primers, and water

- dispense 24,5 µL per well in a plate, or tubes

- pick your colony with a sterilized stick to take some biomass (very few, not the whole colony)

- spread the stick on the bottom of your well/tube, a few seconds, or you will absorb your mix with the stick like a sponge.

- cook it the needed time in a thermocycler and voilà.

I used it on colonies grown on both rich and mineral medium, and it worked for qualitative detection (presence/absence of a functional gene).

I hope it will help you.

Best regards. Emmanuel DUVAL.

I do for E.coli, and suppose that will work for all bacteria/fungy:

pick colony with a tip into 1.5 ml tube with 200 ul of LB-solution plus Ampicillin, 30-60 min grown at +37 with shaking;

aliquot 2 ul in PCR tube for 20 ul reaction mix. PCR condition - standard for PCR:

95 -2 min;

25x cycles:

95 - 20 sec

60 - 20 sec

72 - 30-60 sec

Hi,

I've only done colony PCR on E. coli, but slightly differently to what you describe. My protocol: Pick a colony using a sterile instrument (I use a p200 tip) and suspend in 50uL of colony PCR buffer (Tris-EDTA pH 8.0, 0.1% Triton x-100), boil for 5 min and centrifuge for 5 min at 20,800g to pellet cell debris. Use around 2uL of the lysate for a 30uL PCR reaction. Works every time for me.