Conjugation: Difference between revisions
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Back [[http://openwetware.org/wiki/De_la_Cruz:Resources_%26_protocols]] | |||
== Conjugation (on solid surfaces) == | == Conjugation (on solid surfaces) == | ||
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* Some plasmids require exponentially growing cultures to mate: add the following step: after step 1 dilute cells 1/20 in LB-broth and let them grow at 37ºC and orbital shaking. Check every 1/2 hour the OD<sub>600</sub> and collect the cells when the culture reaches 0.6 aprox. | * Some plasmids require exponentially growing cultures to mate: add the following step: after step 1 dilute cells 1/20 in LB-broth and let them grow at 37ºC and orbital shaking. Check every 1/2 hour the OD<sub>600</sub> and collect the cells when the culture reaches 0.6 aprox. | ||
* Mating on 0.22 μM filters is not essential: you can conjugate directly on the LB agar plate, but then it could be difficult to resuspend the cells. The best way to do this is to add directly to the plate 2 ml of LB broth after conjugation. Add some glass beads to the plate and shake gently to pull out the cells from the LB-Agar surface. Collect the liquid, put it into an assay tube and make the dilutions. | |||
== How to measure conjugation efficiency == | |||
Conjugation is usually measured in terms of conjugation frequency per donor or per recipient. Count the CFU (colony forming units) of donors/ recipients and transconjugants from the dilution plates. Count CFUs in plates that yield in the order of hundreds of colonies. Make AT LEAST three replicates and express conjugation frequency as a median and standard deviation. Notice that since conjugation frequency tends to be Log-normally distributed we do not use the mean. Alternativelly, instead of using the median, you can calculate the mean of the Log distribution. | |||
Latest revision as of 09:06, 7 March 2008
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Conjugation (on solid surfaces)
1.-Grow donor and recipient bacteria in LB broth with the appropiate antibiotics (overnight)
2.-Wash donor and recipient cultures in LB broth to remove antibiotics
3.-Mix 100 μL of donors + 100 μL recipients in an eppendorf tube (For a donor/recipient ratio of 1/1).
4.-Centrifuge briefly and discard supernatant.
5.-Resuspend the cell pellet in aprox. 10 μL of LB broth.
6.-Put a 0.22 μM filter (i.e Millipore filters) on top of a LB-Agar plate. Drop the conjugation mix on the filter.
7.-Incubate for 1 h (repetitive conjugation times are important) at 37ºC
8.-Pick the filter from the plate with sterilized tweezers and put it into an assay tube with 2 ml of LB broth
9.-Vortex to resuspend the cells
10.-Make serial dilutions in LB broth. Plate in selective antibiotics for donors, recipients and transconjugants. We usually determine donor number by plating 10-4 10-5 and 10-6 dilutions. For transconjugants we routinely plate all dilutions (from 1 to 10-6 ).
Modifications and tips
- Some plasmids require exponentially growing cultures to mate: add the following step: after step 1 dilute cells 1/20 in LB-broth and let them grow at 37ºC and orbital shaking. Check every 1/2 hour the OD600 and collect the cells when the culture reaches 0.6 aprox.
- Mating on 0.22 μM filters is not essential: you can conjugate directly on the LB agar plate, but then it could be difficult to resuspend the cells. The best way to do this is to add directly to the plate 2 ml of LB broth after conjugation. Add some glass beads to the plate and shake gently to pull out the cells from the LB-Agar surface. Collect the liquid, put it into an assay tube and make the dilutions.
How to measure conjugation efficiency
Conjugation is usually measured in terms of conjugation frequency per donor or per recipient. Count the CFU (colony forming units) of donors/ recipients and transconjugants from the dilution plates. Count CFUs in plates that yield in the order of hundreds of colonies. Make AT LEAST three replicates and express conjugation frequency as a median and standard deviation. Notice that since conjugation frequency tends to be Log-normally distributed we do not use the mean. Alternativelly, instead of using the median, you can calculate the mean of the Log distribution.
