11 replies to this topic

[biologist1]

Hi!
I am doing colony PCR for an enzyme detection in bacteria but am having problem with its optimization. My results are not consistent. Probably there is some problem with my DNA extraction. I am doing extraction by boiling the bacterial suspension at 99OC for 20 min.After that i centrifuge it at 13000rpm for 10min. On doing gel electrophoresis of the extracted DNA(to look for proper extraction), i never got proper bands rather shining in the wells often. Please guide me if any body has worked on the same lines.
thanx

[phage434]

That doesn't sound like colony PCR, but rather a dirty miniprep.  If you are doing this in preparation for PCR, then you are doing far too much work, and can simply use very small amounts of the colony or culture directly in the PCR reaction, possibly extending the initial 95C step to 5 minutes.  I'd recommend diluting the picked colony or culture 100:1 in water, then using 0.5 ul of this in as a PCR template.

[HomeBrew]

phage434, on 02 January 2011 - 02:26 PM, said:

...can simply use very small amounts of the colony or culture directly in the PCR reaction, possibly extending the initial 95C step to 5 minutes.  I'd recommend diluting the picked colony or culture 100:1 in water, then using 0.5 ul of this in as a PCR template.

This is how I do it as well.

[biologist1]

HomeBrew, on 02 January 2011 - 02:29 PM, said:

phage434, on 02 January 2011 - 02:26 PM, said:

...can simply use very small amounts of the colony or culture directly in the PCR reaction, possibly extending the initial 95C step to 5 minutes.  I'd recommend diluting the picked colony or culture 100:1 in water, then using 0.5 ul of this in as a PCR template.

This is how I do it as well.

What exactly the 100:1 dilution implies? How should I go about it?

[HomeBrew]

Take a small amount of your colony (just a bit on the end of a pipet tip) and resuspend it thoroughly in 50 ul - 100 ul of sterile water.  Use 0.5 ul - 1 ul of this suspension as template in your PCR reaction.  You can freeze the rest if you'll need subsequent samples.

Remember, with colony PCR you can easily have too much colony, but it's pretty hard to have too little -- this is not a case where more is better...

Also if your colonies are transformants from a ligation reaction, be sure to pick them off the original transformation plate, patch them to a fresh plate, and allow them to grow overnight before using as above.  This step cuts down dramatically on false positives.

[phage434]

I take a sterile 2 ul pipet tip, touch the colony, then swirl it in 50 ul of pure water held in a sterile 96 well plate.  I use the same tip to put 2 ul of the water on an index plate to preserve the colony.  If I'm in a hurry, i also inoculate a 10 ml overnight culture with the same tip, in preparation for a glycerol and miniprep, if the colony is correct.  For the PCR, I use a multi-channel pipet to put 0.5 ul into a 10 ul pcr reaction.  If you have a great many, then you can use 150 mm petri dishes and use the multiwell pipet to create the index plate.  I usually try hard to make sure I don't have to search very much for the correct clone by switching antibiotic resistance during the cloning reactions.

[biologist1]

phage434, on 03 January 2011 - 03:18 PM, said:

I take a sterile 2 ul pipet tip, touch the colony, then swirl it in 50 ul of pure water held in a sterile 96 well plate.  I use the same tip to put 2 ul of the water on an index plate to preserve the colony.  If I'm in a hurry, i also inoculate a 10 ml overnight culture with the same tip, in preparation for a glycerol and miniprep, if the colony is correct.  For the PCR, I use a multi-channel pipet to put 0.5 ul into a 10 ul pcr reaction.  If you have a great many, then you can use 150 mm petri dishes and use the multiwell pipet to create the index plate.  I usually try hard to make sure I don't have to search very much for the correct clone by switching antibiotic resistance during the cloning reactions.

Thank you for your response.
I ve tried by using 10ul and even 2ul of the DNA template.
Got results with 10ul template a few times but never with 2ul.
Today i tried the PCR by directly emulsifying the colonies(about 5 colonies in 50ul PCR mix)in the PCR MIX. I increased the denaturation temperature from 3min to 5min at 95OC. Annealing was done at 55OC for 30 cycles.But got no bands.

[HomeBrew]

As I said above, colony PCR is not a case where more is better -- there are PCR inhibitors carried over with the colony.  For PCR to work, you only need vanishingly small amounts of DNA template present...  Does your PCR work on genomic DNA you've isolated from a culture?  How do you know your primers work?

[Adrian K]

Hi there, I also do the same like HomeBrew and phage434.

IF you want to do a 25ul reaction which consist of 2~5ul template, try suspend your single colony in any amount of sterile water.
For me, I usually suspend in 100ul of water into the McFarland standard of 0.5, rough estimate. from here I do a 10 & 100 dilutions and use it as a template.
Usually the template will work in one of the dilutions.

For my experience with P. aeruginosa, the pcr inhibitory is higher and I suspect it might due to the tendency for forming biofilm and its secretory products. You really need to use a very fresh culture, ~24 hr cultures where you hardly sees the green pigmentation, and remember to vortex your suspended sample hard and long enough ~40seconds , inside bio-safety hood. take the top layer of the suspension and do dilutions with it. After you had done a few times, you will know roughly the concentration to start with.

As HomeBrew mentioned, more is not better.

just my 2 cents.

[biologist1]

adrian kohsf, on 06 January 2011 - 04:15 PM, said:

Hi there, I also do the same like HomeBrew and phage434.

IF you want to do a 25ul reaction which consist of 2~5ul template, try suspend your single colony in any amount of sterile water.
For me, I usually suspend in 100ul of water into the McFarland standard of 0.5, rough estimate. from here I do a 10 & 100 dilutions and use it as a template.
Usually the template will work in one of the dilutions.

For my experience with P. aeruginosa, the pcr inhibitory is higher and I suspect it might due to the tendency for forming biofilm and its secretory products. You really need to use a very fresh culture, ~24 hr cultures where you hardly sees the green pigmentation, and remember to vortex your suspended sample hard and long enough ~40seconds , inside bio-safety hood. take the top layer of the suspension and do dilutions with it. After you had done a few times, you will know roughly the concentration to start with.

As HomeBrew mentioned, more is not better.

just my 2 cents.

[biologist1]

thank for your suggestions.
you have explained in good detail.
presently i am thinking of two possibilities for my inconsistent results. first is that my primers might have been mishandled. secondly problem with my template.
do u think DMSO might help me in better results?could you guide me in its concentration per 25ul reaction mixture?
I ve been sub-culturing the same isolate every day for the past one and a half months to use the fresh sub-culture everyday. May be you are right. I think one of the options can be if i do a fresh sub-culture from my preserved stock of the bacterial isolate.
Can we be sure about the amount of DNA in the diluted McFarland suspension e.g. by optical density determination?

[Adrian K]

I use DMSO in almost all of my PCR at 3-5% concentration. But my main organism is B. pseudomallei and not P. aeruginosa.
I use P. aeruginosa in some of my controlled test and I also had cloned some genes from it.
I would suggest you to purify the DNA (using column or kits or any reliable established methods) as a control template for your PCR. Optimize your PCR with the purified DNA. Once you had done this, try run your colony suspensions sample.

For my application, I do not require to know the exact amount of DNA. You can do rough estimations of the amount of bacteria by comparing with McFarland standard, for the colony suspension method you use, you did not extract the dna out from the cells, and I don't think is appropriate to measure the amount of DNA.

And, please check your culture whether your culture is pure. Do API20NE test to confirm identity and/or 16s. I used to be given B. cepacia isolates which was handled down for "generations", I tried my primers and it doesn't work out for many isolates, and my boss blame me for my bad technique. After numerous failures, I decided to check the identity by using 16s and I found that most of the B. cepacia isolates given to me was actually bacillus sp and my primers actually did not failed after all. So, please double or triple confirm your isolates before you start, I learn it the hard way.

Maybe you can share your applications or what you intend to do here, so we can discuss a little further.