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Team:Cambridge-JIC/Guide/Transformation/TransformingAgro
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=Transforming Agrobacteria= | =Transforming Agrobacteria= | ||
There are several protocols for transforming [[Team:Cambridge-JIC/Guide/Materials/PreparingElectrocompetentAgro| electrocompetent Agrobacteria]]. Most standard electroporators work well - but they will need to be suitable for bacterial electroporation, and be able to generate approximately 10kV/cm. | There are several protocols for transforming [[Team:Cambridge-JIC/Guide/Materials/PreparingElectrocompetentAgro| electrocompetent Agrobacteria]]. Most standard electroporators work well - but they will need to be suitable for bacterial electroporation, and be able to generate approximately 10kV/cm. | ||
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The general protocol is as follows: | The general protocol is as follows: | ||
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1. Chill cuvettes and cuvette holder | 1. Chill cuvettes and cuvette holder | ||
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4. Short pulse at 2.5kV | 4. Short pulse at 2.5kV | ||
| - | 5. Immediately add 1ml SOC or LB. | + | 5. '''Immediately''' add 1ml SOC or LB. |
''This has to be done within seconds, otherwise transformation efficiencies start to fall.'' | ''This has to be done within seconds, otherwise transformation efficiencies start to fall.'' | ||
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It's worth bearing in mind that different labs have different protocols for electroporation, and it is best to follow one that has worked with the specific equipment you have. | It's worth bearing in mind that different labs have different protocols for electroporation, and it is best to follow one that has worked with the specific equipment you have. | ||
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Latest revision as of 15:35, 17 October 2014
Transforming Agrobacteria
There are several protocols for transforming electrocompetent Agrobacteria. Most standard electroporators work well - but they will need to be suitable for bacterial electroporation, and be able to generate approximately 10kV/cm.
It is best to check if there is an in house protocol for the exact apparatus you are using. If not, here is one that has worked with strain GV2260 and BioRad GenePulser
The general protocol is as follows:
1. Chill cuvettes and cuvette holder
It's very important for everything to be very cold. Work near a freezer if possible, and avoid removing cuvette from freezer until it's needed. If this is not possible, keep cuvettes on ice.
2. Thaw agrobacteria on ice
Only use freshly thawed cells
3. Add 50ul agrobacteria and 20ng DNA to a 2mm cuvette
To avoid disturbing the salt concentration, it is wise to use DNA at ~100ng/ul in concentration
4. Short pulse at 2.5kV
5. Immediately add 1ml SOC or LB.
This has to be done within seconds, otherwise transformation efficiencies start to fall.
6. Recover for 2.5-3 hours at 20-30 degrees with agitation
Agrobacteria, being a soil bacteria, grows better at these temperatures. 120 rpm is sufficient
7. Plate on LB agar plates, containing antibiotics for strain, and for each of the two Agrobacterium vectors
100ul is usually sufficient.
8. Incubate plates at 30 degrees, and wait. Agrobacteria grow slowly: colonies are first ready after 2.5-3 days.
It's worth bearing in mind that different labs have different protocols for electroporation, and it is best to follow one that has worked with the specific equipment you have.
