I found a protocol for colony PCR in the book 'Mycobacteria protocols':
"Colony PCR is a quick method to check for DCOs. A loopful of cells is resuspended in 400 uL sterile distilled water and vortexed vigorously for 3 min. The cells are killed by heating for 6 min at 100 cels degree and then passed through an 0.2-mm syringe filter for removal from the category 3 laboratory (in the case of M. tuberculosis). The maximum amount of the cell filtrate should be used in a PCR reaction to check for the deletion.“
I tried this protocol on M.smegmatis and M.bovis BCG, but failed to isolate the genome DNA which is enough for PCR test. I decreased the water volume from 400ul to 150ul, I also froze the bacterial suspension for 5 min in -80C before I incubated it at 100C.
there are some issues confusing me, like how much is ' a loopful'? and what is the purpose to do the vortex?
Hi Wenxi,
I think a loopful is referring to a typical inoculation loop used to streak suspensions for single colonies. The volume of material may be ~20ul. I think the vortexing is primarily to break up the clump of cells so the DNA can be released more effectively. Resuspension in water (hypotonic) and boiling may help break the cells open. This simple prep can work but may require optimizing the dilution so that inhibitors are sufficiently dilute but template is adequate. For routine screening most in our lab have moved to the following prep using Biorad instagene matrix which depletes inhibitors:
author: Hannah Ratner
10/10/12
DNA Prep using Biorad Instagene Matrix
For screening possible positive transductants (individual samples in the BSL2):
For picking colonies:
1. Using a sterile wooden stick, transfer each colony into 1 mL of media.
2. Incubate shaking at 37oC for ~ 2 weeks.
For colony DNA extraction:
1. On the morning of the DNA extraction:
a. Pre-heat preheat the convection oven in BSL3 small anteroom to 96-100oC.
b. Prepare a 0.001 % TritonX-100 solution (keep at 4 oC)
c. Add 2.5 mL of the 0.001% TritonX-100 solution to an unopened 25 mL bottle of BioRad Matrix. Mix well and label that Triton X has been added.
2. Re-suspend culture from the picked colony. Save aliquot(s) to store as stocks.
3. Spin down 1 ml of remaining culture in 14 ml conical tube: 4000 rpm, 10 min, RT.
4. Remove supernatant. Re-suspend pellet in 0.1 mL of Biorad Instagene Matrix (mix well before using).
5. Transfer to convection oven. The oven temperature must be at 95oC prior to incubating the sample.
6. Incubate the tube: 95oC, 1 hour.
7. Remove tube to BSL2.
8. Spin sample: 4000 rpm, 20 min, RT.
9. Freeze sample at -80 oC or use immediately for knockout confirmation PCR reactions.
Note: DNA is in the supernatant. Be careful to use only the supernatant in confirmation PCRs, as the pellet will interfere with the PCR reaction. When using frozen DNA prep samples, always thaw and spin for 10 min, 4000 rpm before using in confirmation PCRs.
Best regards,
Brian Weinrick
I agree with Brian. It might be helpful for DNA release from bacteria by adding detergent (Triton or NP40) into lysis buffer. A low concentration of detergent won't inhibit your PCR polymerase.
Good luck!
Hi Brian Weinrick, thank you so much for your answer!
you mentioned one thing I did not pay attention to, the inhibitor in the isolated genome DNA solution. In my failed PCR, I used maximum genome DNA solution in the PCR reaction following the protocol. before I read your answer, I thought the failure resulted from the low concentration of genome DNA, but it may also result from the inhibitor, so I will try to decrease the genome DNA solution first, and see how the PCR will work or not.
Hi guys, Here comes a little update. I tried the colony PCR again with the same genome DNA solution. At this time, I did not add the maximum genome DNA into the PCR reaction, instead, I diluted it and add 1/2 or 1/4 of the former genome DNA amount into each reaction. and the '1/2' reaction worked. I think in my case, the inhibitor concentration does inhibit the PCR efficacy. Thank you Dr. Weinrick!
Hi Dr. Hadir Ahmed Said Okasha, thank you for your advise! could you add a little more detail about the SDS reagent you used in the second method? have you tried a shorter heating time? I just PCR test the genome DNA isolated from the colony of BCG, and it shows 10 mins incubation at 100 can release genome DNA from bacteria into the solution.
I froze the bacteria in 150ul milliQ water at -80 for 5 min, before the 100C incubation.
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