Team:Newcastle/Restriction digests

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=Restriction digestion=
=Restriction digestion=
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To cut DNA at specific base sequences using restriction enzymes  
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==Materials required==
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* Eppendorf tubes
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* Pipettes
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* Appropriate DNA/plasmid
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* Appropriate restriction enzymes
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* 10X buffer
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* Water
==Procedures==
==Procedures==
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# Make up 20 µl reaction mix as follows:
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Add the following as mentioned below to make up to a final volume of 20 µl of reaction mix:
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#*15 µl of DNA/plasmid
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# 15 µl of DNA/plasmid
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#*1 µl of restriction enzyme 1
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# 1 µl of restriction enzyme 1
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#*1 µl of restriction enzyme 2
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# 1 µl of restriction enzyme 2
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#*2 µl of 10X buffer
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# 2 µl of 10X buffer
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#*1 µl of water
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# 1 µl of water
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# Incubate at 37°C for 3 hours
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Incubate the digestion mixture at 37°C for 3 hours
==Notes==
==Notes==
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*No more than 10% of enzyme - solution contains glycerol which inhibits reaction  
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*No more than 10% of enzyme should be used for a single reaction - glycerol inhibits reaction  
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*10x buffer must be diluted to 1x i.e. 10% final volume  
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*10x buffer must be diluted to 1x i.e. 10% final volume.
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Latest revision as of 15:54, 5 August 2010

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Contents

Restriction digestion

Materials required

  • Eppendorf tubes
  • Pipettes
  • Appropriate DNA/plasmid
  • Appropriate restriction enzymes
  • 10X buffer
  • Water

Procedures

Add the following as mentioned below to make up to a final volume of 20 µl of reaction mix:

  1. 15 µl of DNA/plasmid
  2. 1 µl of restriction enzyme 1
  3. 1 µl of restriction enzyme 2
  4. 2 µl of 10X buffer
  5. 1 µl of water

Incubate the digestion mixture at 37°C for 3 hours

Notes

  • No more than 10% of enzyme should be used for a single reaction - glycerol inhibits reaction
  • 10x buffer must be diluted to 1x i.e. 10% final volume.


Go back to our Protocol List

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