Team:Lyon-INSA-ENS/Realisation/Protocols/Fermentas

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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"> Fermentas digestion </font> </p>
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This protocol aims at inserting plasmids notably into NM522 strains, or any other E.Coli strain
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This protocol aims at cutting plasmids on specific restriction sites using restriction enzymes.  
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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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<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> In an eppendorf tube, add the following solutions in any order :<br/>
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-250 ng DNA ( if the concentration is unknown, use 5µL )<br/>
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-2.5µL (1X) or 5µL (2X) 10X tango buffer ( this depends on the enzymes used )<br/>
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-water to get a volume of 25µL after addition of the enzymes
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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> Add 1µL of each one of the desired enzymes.
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<b>3.</b> Incubate on ice for 10mn
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<b>3.</b> Incubate at 37°C for 1h30
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<b>4.</b> Centrifuge at 4°C, 10mn, 5000rpm
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<b>4.</b> Incubate at 70°C for 10 mn to inactivate the restriction enzymes.
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<b>5.</b> Empty the supernatant
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<b>6.</b> Add V/2 mL cold and sterile CaCl2 100mM
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<b>7.</b> Incubate for 20mn on ice
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<b>8.</b> Centrifuge at 4°C, 5mn, 5000rpm
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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>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>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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Latest revision as of 10:19, 10 January 2012





Fermentas digestion





This protocol aims at cutting plasmids on specific restriction sites using restriction enzymes.





Procedure



1. In an eppendorf tube, add the following solutions in any order :
-250 ng DNA ( if the concentration is unknown, use 5µL )
-2.5µL (1X) or 5µL (2X) 10X tango buffer ( this depends on the enzymes used )
-water to get a volume of 25µL after addition of the enzymes


2. Add 1µL of each one of the desired enzymes.


3. Incubate at 37°C for 1h30


4. Incubate at 70°C for 10 mn to inactivate the restriction enzymes.








ENS assystem Biomérieux INSA INSA
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