Team:University College London/Protocols/RestrictionEnzymeDigest1

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== Enzyme Digest ==
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== Enzyme Digest Protocol 1 ==</noinclude>
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</noinclude>
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== All Hail Rhiannon the Wiki Mistress ==
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'''Step 1 - Thawing cells:''' Thaw all materials on ice
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1. Thaw all materials on ice
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2. Add the following ingredients to autoclaved/sterile eppendorf tubes  
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'''Step 2 - Adding Ingredient:''' Add the following ingredients to autoclaved/sterile eppendorf tubes  
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Insert Table
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{| class="wikitable"
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|-
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! Component !! Amount (ul) (one enzyme used) !! Amount (ul) (two enzymes used)
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|-
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| dH20 || 2.5 || 1.5
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|-
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| Buffer 1x || 1 || 1
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|-
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| DNA template|| 5|| 5
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|-
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| BSA || 0.5|| 0.5
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|-
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| Enzyme 1 || 1 || 2
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|-
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| Enzyme 2 || N/A|| 1
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|}
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3.Flick contents gently and centrifuge.
 
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4.To centrifuge, add sample eppendorf + balancer (fill another tube with equal volume of water if only have one sample) and screw lid on tight
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'''Step 3 - Addition of BioBrick:''' Flick contents gently and centrifuge.
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5. Then run on the following conditions (by turning the dial)
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'''Step 4 - Centrifuge:'''
RPM: 14000
RPM: 14000
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Temperature: 18oC
Temperature: 18oC
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'''Step 5 - Digest Program:''' Place the samples on a thermocycler under the following conditions:
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RPM: 550
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Time: 2.5 hours
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Temperature: 37oC
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'''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:
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RPM: 550
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Time: 25 minutes
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Temperature: 65oC
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<noinclude>
<noinclude>
{{:Team:University_College_London/templates/foot}}
{{:Team:University_College_London/templates/foot}}
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</noinclude>

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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