Problem with colony PCR - MBL detection in Pseudomonas aerugfinosa (Jan/02/2011 )
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
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.
phage434 on Sun Jan 2 22:26:44 2011 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.
HomeBrew on Sun Jan 2 22:29:53 2011 said:
phage434 on Sun Jan 2 22:26:44 2011 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?
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.
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.
phage434 on Mon Jan 3 23:18:41 2011 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.
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?
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.
adrian kohsf on Fri Jan 7 00:15:10 2011 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.