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 more efficient than the CaCl2 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> Take x mL from the previous culture and centrifuge at .
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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>10.</b> Incubate for 1h at 37°C.
<b>10.</b> Incubate for 1h 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