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| - | {{:Team:DTU-Denmark/Templates/Standard_page_begin|Transformation and selection}}
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| - | == Part 1: Transformation by electroporation ==
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| - | Materials:
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| - | * ice
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| - | * test-tubes
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| - | * cuvettes
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| - | * recovery media ( LB medium) (check for contaminations!)
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| - | * your strain
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| - | * DNA
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| - | Procedure:
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| - | <ol style="list-style-type:decimal">
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| - | <li> Get competent cells and store them on ice (the number on the lid indicate the number of transformation the amount corresponds to).
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| - | <li> Pool the competent cells into one tube when they’re thrown (takes ~ 5min). This is done to ensure a homogen batch.
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| - | <li> Put cuvettes for electroporation on ice.
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| - | <li> Label as many glass tubes and cuvettes as samples and in addition to that, prepare some extra tubes and make sure to include a negative control (competent cells that are not transformed). Add 1 ml recovery media (LB) to your tubes with glass pipette and have them ready just right after the electroporation. The LB media MUST at least have room temperature in order for the cells to survive. Check the LB media by shaking to ensure that it is sterile and there’s no bacteria growth.
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| - | <li> Electorporation (in room 217)
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| - | <ol style="list-style-type:lower-alpha">
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| - | <li> Use program EC2, and set the form to ‘time ms’, press the right blue square bottom to get started.
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| - | <li> Add 2 μl of the ligation (DNA) to one side of the cuvette( usually we use the side with apophysis to recognize and remember easily).
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| - | <li> Add 40 μl of the competent cells to the side of cuvette which we put DNA before.
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| - | <li> Mix by tapping, this ensures that all liquid is at the bottom and that there’s no bubble, dry the cuvette. Don’t touch the metal on the cuvettes.
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| - | <li> Pipet 1 ml recovery media.
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| - | <li> Place the cuvette in the machine and pulse (press the pulse bottom), write down the time( usually is above 5 and below 5.8)
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| - | <li> Immediately add 1 ml recovery media to the cuvette, pipette gently up and down for 1-2 times and try to get as much of the liquid retransferred to the tube where you tookrecovery media from. (incline the cuvette to get as much liquid as we can). Important: half of the cells are lost every 15 sec until medium is added.
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| - | <li> Keep track of the time constant, it should preferentially be around 5.60. Mark the tube if the time constant is below 5.00 as the cells might be dead/weak. If the time-constant is 0.00 (ARC) the cells are dead! Try again, maybe with less ligation mix.
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| - | </ol>
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| - | </ol>
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| - | * ARC can be due to different factors:
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| - | <ol style="list-style-type:lower-roman">
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| - | <li> Too much DNA
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| - | <li> Too many ions
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| - | <li> Bubbles
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| - | <li> Liquid on the outside of the cuvette (i.e. the cuvette was not dried properly)
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| - | == Part 2: Selection of transformants ==
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| - | Materials:
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| - | * antibiotic plates
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| - | * LB plates
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| - | Procedure:
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| - | # You will be plating 100 ul on each plate (with antibiotic corresponding to the selection marker on the plasmid), e.g. with “Sebastiens technique”.
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| - | # First, decide if you need to make a dilution series which depends on how well transformation goes. If it is high, then without diluting there will be an over-grow of bacteria on the plate and you can’t select a colony from there.
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| - | # Choose your dilutions, e.g. from 100 to10-7 in order to get an appropriate number of bacteria for plating out. High efficiency transformation: use dilution -5, -6 or -7.
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| - | # Plate 100 µL from your 10-X dilutions onto the antibiotic plates and spread (this is our experiment).
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| - | # Negative control: Plate 100 µL from your undiluted bacteria culture (100 dilution)that was not transformed onto the selective plate.
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| - | # Positive control: Plate 100 µL from your 10-x dilution onto a non-selective plate and spread.
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| - | # Incubate your plates overnight at 37ºC.
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| - | # Place the rest of the LB-media with transformed cells on the bench overnight. If colonies are lacking the next day; plate again.
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| - | Good advises
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| - | * Prepare a couple more of the LM-tubes and cuvettes than you have samples – if you by accident kill some of the cells during the electroporation.
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| - | {{:Team:DTU-Denmark/Templates/Standard_page_end}}
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