Team:Lyon-INSA-ENS/Realisation/Protocols/TSS trans

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<p style="text-align : center"> <font color="green" size="5"> CaCl2 chemical transformation </font> </p>
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<p style="text-align : center"> <font color="green" size="5"> TSS chemical transformation </font> </p>
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It has been found less efficient than the TSS transformation protocol, thus we do not recommend using it.
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It has been found more efficient than the CaCl2 transformation protocol, thus we recommend using it.
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<b>1.</b> Monitor the OD600 of a 50mL culture of NM522 cells in LB medium at 37°C. Proceed to the next step when it reaches 0.2-0.4 ( not higher than 0.6 ), which takes approximately 4 hours.
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<b>1.</b> Monitor the OD600 of a 5mL culture of NM522 cells ( or the strain you want to transform ) in LB medium at 37°C. Proceed to the next step when it reaches 0.3-0.4 ( not higher than 0.6 ), which takes approximately 3 hours.
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<b>2.</b> Aliquot the culture into fractions of the desired volume V ( 10 or 15 mL usually ).
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<b>2.</b> Take 1 mL from the previous culture and centrifuge at 6000rpm for 2mn .
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<b>3.</b> Incubate on ice for 10mn
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<b>3.</b> Resuspend into 100µL of TSS. Starting from this step, keep the bacteria on ice.
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<b>4.</b> Centrifuge at 4°C, 10mn, 5000rpm
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<b>4.</b> Incubate on ice for 5-10mn.
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<b>5.</b> Empty the supernatant
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<b>5.</b> Add 5µL of the DNA to be transformed.
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<b>6.</b> Add V/2 mL cold and sterile CaCl2 100mM
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<b>6.</b> Incubate on ice for 10mn.
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<b>7.</b> Incubate for 20mn on ice
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<b>7.</b> Heat shock the bacteria by heating them at 42°C for 50s.
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<b>8.</b> Centrifuge at 4°C, 5mn, 5000rpm
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<b>8.</b> Incubate on ice for 2mn.
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<b>9.</b> Empty the supernatant and resuspend ( do not vortex ) in V/15 mL CaCl2 and V/30 mL glycerol 40%
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<b>9.</b> Add 900µL LB and mix by inverting the tube.
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<b>10.</b> Aliquot the bacteria in 200 µL fractions and keep on ice. These bacteria should be used within the day.
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<b>10.</b> Incubate for 1h at 37°C.
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<b>11.</b> Add the desired quantity of DNA to the fractions. For positive control, we used Puc18 plasmid. For negative control, we used water.
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<b>12.</b> Incubate for 30mn on ice
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<b>13.</b> Heat shock at 42°C for 2mn
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<b>14.</b> Incubate for 5mn on ice
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<b>15.</b> Add 800µL of LB medium and incubate for 1h at 37°C
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<b>16.Optional</b> Concentrate 10X by centrifuging for 2mn at 11000 rpm, eliminate the supernatant and resuspend in 100µL LB.
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<b>17.</b> Plate 100µL of bacteria on LB medium with the appropriate antibiotic resistance and incubate at 37°C.
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<b>11.</b> Plate 200µL on LB medium with the appropriate antibiotic.
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Latest revision as of 10:45, 10 January 2012





TSS chemical transformation





This protocol aims at inserting plasmids notably into NM522 strains, or any other E.Coli strain

It has been found more efficient than the CaCl2 transformation protocol, thus we recommend using it.





Procedure



1. Monitor the OD600 of a 5mL culture of NM522 cells ( or the strain you want to transform ) in LB medium at 37°C. Proceed to the next step when it reaches 0.3-0.4 ( not higher than 0.6 ), which takes approximately 3 hours.


2. Take 1 mL from the previous culture and centrifuge at 6000rpm for 2mn .


3. Resuspend into 100µL of TSS. Starting from this step, keep the bacteria on ice.


4. Incubate on ice for 5-10mn.


5. Add 5µL of the DNA to be transformed.


6. Incubate on ice for 10mn.


7. Heat shock the bacteria by heating them at 42°C for 50s.


8. Incubate on ice for 2mn.


9. Add 900µL LB and mix by inverting the tube.


10. Incubate for 1h at 37°C.


11. Plate 200µL on LB medium with the appropriate antibiotic.








ENS assystem Biomérieux INSA INSA
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