Difference between revisions of "Help:Protocols/Restriction Digest"

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''estimated time: 30 min. active, 50 min. incubation''
 
''estimated time: 30 min. active, 50 min. incubation''
  
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The following protocol assumes you'll be doing restriction digests for 3A assembly, therefore we refer to your digests as:
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The following protocol assumes you are simply doing a restriction digest for quality control, you can use the [[#Single_Reaction|protocol below]].
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*Part A (The 1st part in the future composite part)
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*Part B (The 2nd part in the future composite part)
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*Linearized plasmid backbone (The destination plasmid backbone for your composite part)
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If you are simply doing a restriction digest for quality control, you can use the [[#Single_Reaction|protocol below]].
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If you'll be doing restriction digests for 3A assembly, see the [[Help:Protocols/3A_Assembly|3A assembly protocol]] or [[Help:Protocols/Linearized_Plasmid_Backbones|linearized plasmid backbone protocol]].
  
 
<html><iframe src="http://player.vimeo.com/video/45793760" width="320" height="180" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe> <p><a href="https://igem.org/Videos/Restriction_Digest">Restriction Digest</a> from <a href="https://igem.org/Videos">iGEM Videos</a>.</html>
 
<html><iframe src="http://player.vimeo.com/video/45793760" width="320" height="180" frameborder="0" webkitAllowFullScreen mozallowfullscreen allowFullScreen></iframe> <p><a href="https://igem.org/Videos/Restriction_Digest">Restriction Digest</a> from <a href="https://igem.org/Videos">iGEM Videos</a>.</html>
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*Ice and bucket/container
 
*Ice and bucket/container
 
*(1) 8-tube strip, or (3) 0.6ml thin-walled tubes   
 
*(1) 8-tube strip, or (3) 0.6ml thin-walled tubes   
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*Part A (Purified DNA, > 16ng/ul)
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*BioBrick Part in BioBrick plasmid (Purified DNA, > 16ng/ul)
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*Part B (Purified DNA, > 16ng/ul)
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*Linearized plasmid backbone (25ng/ul)
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*dH2O
 
*dH2O
 
*NEB Buffer 2
 
*NEB Buffer 2
 
*BSA
 
*BSA
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*Restriction Enzymes: EcoRI, SpeI, XbaI, PstI, DpnI
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*Restriction Enzymes: EcoRI, SpeI, XbaI, PstI
 
*Thermal cycler
 
*Thermal cycler
  

Revision as of 17:50, 6 July 2015

Restriction Digest Protocol

estimated time: 30 min. active, 50 min. incubation

The following protocol assumes you are simply doing a restriction digest for quality control, you can use the protocol below.

If you'll be doing restriction digests for 3A assembly, see the 3A assembly protocol or linearized plasmid backbone protocol.

Restriction Digest from iGEM Videos.


Materials

  • Ice and bucket/container
  • (1) 8-tube strip, or (3) 0.6ml thin-walled tubes
  • BioBrick Part in BioBrick plasmid (Purified DNA, > 16ng/ul)
  • dH2O
  • NEB Buffer 2
  • BSA
  • Restriction Enzymes: EcoRI, SpeI, XbaI, PstI
  • Thermal cycler

Notes:

  • You should keep all materials on ice.
  • At iGEM HQ we use restriction enzymes from New England Biolabs


Single Reaction

  1. Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul.
  2. Add 2.5ul of NEBuffer 2.
  3. Add 0.5ul of BSA.
  4. Add 0.5ul of EcoRI.
  5. Add 0.5ul of PstI.
  6. There should be a total volume of 20ul. Mix well and spin down briefly.
  7. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes. We incubate in a thermal cycler with a heated lid
  8. Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.