Denaturing conditions should be selected by considering the thermal cycler model and the type of tube that will be used. The general guideline for standard amplification is to denature at 94°C for 30sec. Since you are doing Colony PCR i suggest you to increase the time of denaturation. Like Robert Shore said 5min at 94-95°C or 2min at 98°C.

If you are using a more thermo resistant enzyme try 98°C (often better to use high temperature and shorter duration) and increase the time up to 2min and observ if the result is better.

Cheers!

Thanks for the input!

I am indeed using a highly thermostable Pfu-taq fusion enzyme, so it sounds like I can go with a 2 min initial denaturation time at 98°C. To hedge my bets, I'm also going to heat my cell suspensions at 95°C for 5 minutes, pellet cellular debris, and use the supernatant as template solution. I'll try both procedures alone, then together to see what the individual and combined effects on my yield are.

But there's a wrinkle: due to the particulars of the assay I am developing, I need to perform colony PCR from liquid culture (I suppose inoculum PCR would be a better term). My first thought is to pellet cells from a volume of culture, wash once with nuclease-free water, resuspend in a volume of NFW, and process this suspension as above: heat at 95°C for 5 minutes, pellet cellular debris, collect supernatant, and use 1 uL supernatant for PCR.

The question then becomes: how much sample to process, and what volume to resuspend it in? It would help it I could get a better sense of how many cells is too many for the purposes of colony PCR. I could start with stationary phase E. coli culture at 2*10^9 cells/mL, make 10-fold serial dilutions, pellet cells from 100 uL samples, wash and resuspend in the same volume NFW, heat at 95°C for 5 minutes, pellet cellular debris, and use 1 uL of supernatant for  perform PCR, and see which ones work. That procedure would assay 2*10^7 to 1 cell/reaction, which should give me a decent colony PCR calibration curve. Sound like a good idea?

Pretty easy to test actually, take your mere liquid culture and perform a geometric dilution. this way you will obtain several different concentration and the one that seems to be the best on the electrophoresis gel is the one you should take. (Strongest band without smear).

Just read that it's what you are planning to do sorry, i think it's a good idea and it will give you the answer you seek.

hi. in hot start 95-98C and 5 minutes

To analyze  cloned sequences, randomly chosen clones are generally tested by PCR-colony. A single colony  isolated and added into 5 μL sterile water is used as template DNA in PCR reaction. The amplification program consists genetrally in : a pre-denaturation at 94 °C for 4 min followed by 25-30 cycles of denaturating at 94 °C for 1 min, annealing at 50 °C for 1 min, extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min.

After electrophoresis, an agarose gel is stained and DNA bands are compared to the expected sizes to determine positive clones.Additionally, these clones should be sequenced to confirm the right orientation of the DNA insertion. 

Good luck!

Hi

Initial denaturation at 95°C for 2 minutes is recommended prior to PCR cycling to fully denature the DNA. Avoid longer or higher temperature incubations (unless required due to high-GC content of template)

Hallo Brendan, I dont use a phusion polymerase for colony pcr, just a simple taq-polymerase. I use a phusion polymerase to amplify fragments were it is important to have no errors. But to check which of the colonies contain the correct fragment, a simple taq polymerase is enough. The phusion is much more expensive than the taq polymerase. I´m doing the following procedure: To reduce the costs I use only 12,5 µL PCR reaction mix per PCR tube. First I prepare a master mix containing the follwoing components:

1. steril water: 7.375 µL

2. 10 X Reaction buffer: 1.25 µL

3. 2 mM dNTP´s (each): 1.25 µL

4. primer 1 (5 mM): 1.25 µL

5. primer 2 (5mM): 1.25 µL

6. Taq Poly (5U/µL): 0.125 µL  = 0.7 U/per reaction

I prepare the number of colonies plus one for 10, 2 for 20 reaction tubes and so on  to have enough due to pippeting mistakes.

Then I pipett 12.5 µL into each tube. Take the pipette pick a just a few amount of colony from the plate and resuspend the colony within the PCR tube containing the reaction mix. Then I use the following PCR cycle steps.

1. 94 °C --> 4 min

2. 94 °C --> 30 sec.

3. Annealing temp of the primer: 30 sec.

4. Extension: 72 °C  (1min/kb)

5. Final extension: 72°C --> 5 min

6. Hold: 4 °C.

Thats it. No further steps and it works quite well and is cheap. Alternatively you can resupsend the colony in 20 µL PEG (if you need a protocol, I can send it), wait 10 min and take 1 µL per tube. For liquid cultures, I centrifuged 1 mL cell culture, discard the supernatant and resuspend the pellet in 100µL steril water, heat up for 5 min at 98 °C, centrifuge again and used 1 - 2 µL from the supernatant for the PCR.  

Thanks for the suggestion Marco! I would leverage taq for cost savings if I could, however fidelity is quite important to my protocol. I will be using liquid cultures, and I like the protocol that you suggested for processing samples for that purpose. However, for the purposes of my assay, a 100-fold dilution would be preferable to 1000-fold, so I'm going to centrifuge 100-uL of culture to pellet cells (5000g 10 min), resuspend in same volume NFW, heat at 98C for 5 minutes, centrifuge to pellet cell debris but not chromosomal DNA, and use 1 uL of supernatant.

This brings up another question: can you recommend centrifuge run parameters to pellet cell debris but not chromosomal DNA? My target sequence is on the host genome, not a plasmid, so I can't use 17000g as is normally used in mini-prep procedures, as this would pellet genomic DNA along with cellular debris.

Which organism are you using??? So I tried to make a kind of colony PCR with clostridium ljungdhalii but i was just able to amplify short fragments in the range of 500 bp. Larger fragments didn´t worked. I think the genomic DNA was fragemented, because when i used a genomic DNA extraction kit it worked.

So, at the moment I work with S. cerev. and I also need to verify positiv gene integration and when I was searching for a protocol I found this one:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182553/

But so far, I never tried it.  Maybe it will help you. Here in this paper, they discared the supernatant and used the resuspended pellet for the PCR!!  Otherwise, maybe a chloroform/phenol extraction might work. But it is quite time consuming.

Yup, looks right: pellet cell debris, discard supernatant. The more that I look into colony PCR, and the more steps I add to my protocol, the more that it resembles a spin-column protocol! Since I have the spin-column kits, I'm going to use them for my first sequencing runs, then play with colony PCR to see if I can lower sample-prep costs for upscaling the assay. Thanks for all of the suggestions everyone!