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Team:Lyon-INSA-ENS/Realisation/Protocols/Fermentas
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<b>1.</b> In an eppendorf tube, add the following solutions in any order :<br/> | <b>1.</b> In an eppendorf tube, add the following solutions in any order :<br/> | ||
| - | -250 ng DNA<br/> | + | -250 ng DNA ( if the concentration is unknown, use 5µL )<br/> |
-2.5µL (1X) or 5µL (2X) 10X tango buffer ( this depends on the enzymes used )<br/> | -2.5µL (1X) or 5µL (2X) 10X tango buffer ( this depends on the enzymes used )<br/> | ||
| - | -water to get a volume of | + | -water to get a volume of 25µL after addition of the enzymes |
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| - | <b>4.</b> Incubate at 70°C for 10 mn | + | <b>4.</b> Incubate at 70°C for 10 mn to inactivate the restriction enzymes. |
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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.
