Team:University College London/Protocols/RestrictionEnzymeDigest1

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Revision as of 09:00, 1 August 2012

Enzyme Digest

Step 1 - Thawing cells: Thaw all materials on ice

Step 2 - Adding Ingredient: Add the following ingredients to autoclaved/sterile eppendorf tubes

Insert Table

Step 3 - Addition of BioBrick: Flick contents gently and centrifuge.

Step 4 - Centrifuge:

RPM: 14000

Time: 1 minute

Temperature: 18oC

Step 5 - Digest Program: Place the samples on a thermocycler under the following conditions:

RPM: 550

Time: 2.5 hours

Temperature: 37oC

Step 6 - Denaturing Enzymes: If you are not running the samples on a gel immediately, denature the restriction enzymes by running the samples on a thermocycler under the following conditions:

RPM: 550

Time: 25 minutes

Temperature: 65oC

@@ Line 12: / Line 12: @@
 '''Step 3 - Addition of BioBrick:''' Flick contents gently and centrifuge.
-'''Step 4 - Centrifuge:''' To centrifuge, add sample eppendorf + balancer (fill another tube with equal volume of water if only have one sample) and screw lid on tight
+'''Step 4 - Centrifuge:'''
-'''Step 5 - Digest Program:''' Then run on the following conditions
 RPM: 14000
@@ Line 21: / Line 19: @@
 Temperature: 18oC
+'''Step 5 - Digest Program:''' Place the samples on a thermocycler under the following conditions:
+RPM: 550
+Time: 2.5 hours
+Temperature: 37oC
+'''Step 6 - Denaturing Enzymes:''' If you are not running the samples on a gel immediately, denature the restriction enzymes by running the samples on a thermocycler under the following conditions:
+RPM: 550
+Time: 25 minutes
+Temperature: 65oC
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