A while ago someone had the same issue as you. He was also boiling the cells directly in the PCR mix. This is what I had to say:

"I prepare a colony PCR lysis buffer (TE + 0.1% Triton-X100) then boil each colony to be screened for 5 minutes in 15 μL of this lysis buffer, then spin down (~13000 G) for 10 mins. I use 2 μL of the supernatant as template for the colony PCR. Never failed me. With that said, I should also say that I patch the colonies to be screened on a new plate, let them grow, and then lyse them. I do this for the sake of organization and, in my case, it prevents false positives (you should do this if you're trying to identify recombinant plasmids)."

It always worked for me and it also solved that researcher's issue. Let us know if it works for you too!

I was using the same technique and haven't had any problems. What I do is to submerge the tip of the toothpick and leave it there for about 10 sec before "washing" it in the PCR cocktail (like you would a spoon when stirring coffee). Also, I try to avoid colonies that are too small. Depending on the primers that you use, no amplification may also mean that there simply is no such fragment in your plasmid. 

Hope it helps :)

Thanks, Wei. Colony PCR for me is really tricky. That is why sometimes I directly do my PCR with the plasmid DNA isolated.

try to add bacterial cell in PCR mix as minimum as you can have. Sometimes we think that the amount is too less and pcr will not work but practically it is opposite to that.

A while ago someone had the same issue as you. He was also boiling the cells directly in the PCR mix. This is what I had to say:

"I prepare a colony PCR lysis buffer (TE + 0.1% Triton-X100) then boil each colony to be screened for 5 minutes in 15 μL of this lysis buffer, then spin down (~13000 G) for 10 mins. I use 2 μL of the supernatant as template for the colony PCR. Never failed me. With that said, I should also say that I patch the colonies to be screened on a new plate, let them grow, and then lyse them. I do this for the sake of organization and, in my case, it prevents false positives (you should do this if you're trying to identify recombinant plasmids)."

It always worked for me and it also solved that researcher's issue. Let us know if it works for you too!

Hassan Ishag,

PhD,

You can also grow selected clones, purify the plasmid with plasmid purification kits and then use the purified plasmid as a template for your PCR.

Manoj, yes I am doing it to put less bacterial cells in the PCR mix. But sometimes, I am doubting myself if I had put enough bacterial cells to the PCR mix so my tendency is to pick more cells. But yes, less is more in doing colony PCR.

Daniel, thanks for the reply. Your answer is really interesting and might want to try it. I did not know that triton X is a good lysis buffer. I am going to update you as soon as I get my PCR result!

Hassan, yes that is my last option if nothing in my colony PCRs worked. I know it is a little expensive and takes a longer time to have the result. But yeah, might be my last option.

hiii, will colony PCR work out for Lactic acid bacteria???

Dear Christian Cantos,

Hassan Ishag,

PhD,

It does not take long time. I do it frequently these days and it only takes 2 days to do it. One day to culture the single clone and next day can extract the plasmid and run the PCR even you can send the sample for sequencing. 

Instead, you can run the extracted plasmid that expected to have the insert along with control plasmid (empty one) in a gel. if your plasmid contains the insert, then you can observe a shift in the band (it runs slower than the empty one). This may help you screen the recmbinant plasmid.

Hassan, if you are screening a lot of colonies for insertion of the fragment. I don't think it is a good idea to isolate plasmid DNA and do the PCR after. That is why, we are doing the colony PCR first in order to have the right clones to grow in liquid for plasmid DNA isolation for further analysis. 

Colony PCR can be tricky. The reason for this is the sometimes poor quality of the colony material (presence of contaminants, genomic DNA, etc.). If you get a negative result from a colony PCR it is often possible that your clone is still positive. It is very important that the colonies you use are freshly grown (that is overnight growth). If possible, always include a positive (e. g. purified plasmid DNA) and a negative control (containing all components except template DNA) in your experimental set-up. A common error is that people use too much colony material. Less is more when it comes to colony PCR.

@ Poorna,

According to this publication (see attached), yes, colony PCR can be used for Lactic acid bacteria. I believe that their PCR results would have been even prominent if they would have used less amount of colony for PCR (they used the whole colony to do PCR). Try and find out.

Good luck.

I agree with the less is better answer of Christian. I usually touch a colony into about 50ul of water and use 1-2 ul of that. With a plasmid carrying strain you are in vast vast excess of template. 

A good easy test could be to run some complicate reactions in triplicate: put the toothpick into the colony, then immerge the toothpick into the first tube, then the same toothpick into the second and the third. Doing that, you operate a serial dilution. If you don't get any amplification in the first tube, but you get it into the second and/or the third, probably the problem is that you have too much bacterial cells in non amplified samples.

With PCR, sometimes the less is the better, with colony-PCR still more, because you're not working with pure DNA and you can have a high concentration of inhibitors. 

Recently I discover a "direct PCR" protocol for fungi: I simply put some fungal cells (once again, the less is the better) into an empty sterile tube, then I put the closed tube into the microwave at the maximum power for 5 mins. After that, I add into the tube 50 ul of TE buffer, I vortex it for few seconds and I centrifuge at 13000 rpm for 5 mins. Then, I use the supernatant as template for PCR.

Sounds weird, but it work and is possible to save a lot of time. Maybe you can do the same with bacteria.

Good luck.

The methods used to uptake the plasmids into competent cells also play a role in it. I have used both (1) heat-shock and (2) electroporation methods to transform same plasmids into regular and electro-graded competent cells respectively, and selected on a selection medium. Colony PCR was then performed. The results had shown that majority of colonies derived from electroporated cells produced prominent bands, while the heat-shock ones had less prominent bands. This may have been to do with how many copies of plasmids was taken in. The good news is that nowadays the electro-grade competent cells can be made in the lab.

Hi Christian, you mention that you get the colony PCR working sometimes meaning that you're doing it great up to this point. In our lab, if I'm taking colonies directly from the blue/white screening plate, I use a 1 ul inoculating loop to transfer the colony onto a streak plate, then submerge the loop into the colony PCR mix afterwards. This will make sure that there isn't too much starting material going into the PCR and if the colony is positive for the insert then you have a pure colony plate ready for mini-prep. Furthermore, if you want to screen many colonies, you can section a plate into 4-8 grids.

If it hasn't been mentioned before, you can always pick a colony and resuspend it in 50-100ul TE buffer and use 1-5ul of that as your template.

Thank you everyone for the comments and suggestions. I think the main issue is to put less bacterial cells either by diluting it in buffer or with triton X or touch as less as possible the coloy before putting it in the PCR mix. One suggestion also is to make a loop of the bacterial cells, submerge it to 50-100uL TE buffer and then boil it at 95C for 10 minutes, spin it down and use 2uL of the supernantant which I followed and it worked pretty well. 

If you work with E.coli, then you can use toothpick inoculate your colony into 100ul medium (LB with antibiotic) then incubate for 2 hours at 37 degree. You can directly use 2 ul of cultured for PCR. 

 Fresh and diluted colony usually may cause better results..

Good Luck 

Thank you for the suggestion Payal!

Colonies containing recombinant vector DNA were identified using colony PCR screening. Several colonies were randomly picked by sterile toothpick and each resuspended in 10 μl water, then the wet toothpick was streaked on LB agar containing appropriate antibiotic and allowed to grow at 37 °C overnight. The remainder of each suspension was heated at 99 °C for 5 minutes before cell debris was removed by centrifugation. 

As most of you have different ways of doing colony PCR, I will tell you a very simple way of doing colony PCR while parallelly you can patch colonies.

Colony PCR protocol:

1. I make master mix and use 20 ul of master mix per colony.

2. Scrape half of the colony and first patch on respective plate and throw the tip into the tube containing PCR mix. Stir the master mix with the pipette. I know you may be wondering if enough cells went in (to answer your question take another PCR tube filled with master mix and compare them, the tube with master will be clear and the tube with the suspended cells will be a bit turbid. There will be so many cells to make up a single visible colony and therefore still the will be so may cells in the tip after patching) 

3. Continue to the PCR (the first denaturation step at 95 C or 94 C is expected to burst open the cells and release DNA into the tube)

This protocol never failed me until I am using older colonies.

The colonies should not be older than over night grown (If I do transformation today the next day morning I will plan my colony PCR, if you want to store the transformed plate don't forget to patch the cells before using them for colony PCR)

Thank you Daniel Lorenz Winter for the protocol.

It worked Beautifully.

Regards

Arman

it is possible to use random primers for amplification of genomic DNA by using colony PCR procedure for any organism