Team:University College London/Protocols/RestrictionEnzymeDigest1

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(Difference between revisions)
(→Enzyme Digest Protocol 1)
 
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'''Step 2 - Adding Ingredient:''' Add the following ingredients to autoclaved/sterile eppendorf tubes  
'''Step 2 - Adding Ingredient:''' Add the following ingredients to autoclaved/sterile eppendorf tubes  
-
Insert Table
+
{| class="wikitable"
 +
|-
 +
! Component !! Amount (ul) (one enzyme used) !! Amount (ul) (two enzymes used)
 +
|-
 +
| dH20 || 2.5 || 1.5
 +
|-
 +
| Buffer 1x || 1 || 1
 +
|-
 +
| DNA template|| 5|| 5
 +
|-
 +
| BSA || 0.5|| 0.5
 +
|-
 +
| Enzyme 1 || 1 || 2
 +
|-
 +
| Enzyme 2 || N/A|| 1
 +
|}
 +
 
'''Step 3 - Addition of BioBrick:''' Flick contents gently and centrifuge.  
'''Step 3 - Addition of BioBrick:''' Flick contents gently and centrifuge.  

Latest revision as of 01:36, 27 September 2012

Enzyme Digest Protocol 1

Step 1 - Thawing cells: Thaw all materials on ice

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

Component Amount (ul) (one enzyme used) Amount (ul) (two enzymes used)
dH20 2.5 1.5
Buffer 1x 1 1
DNA template 5 5
BSA 0.5 0.5
Enzyme 1 1 2
Enzyme 2 N/A 1


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


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