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

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<td><div id="splash-title">Restriction Digests</div></td>
 
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When using parts for 3A assembly, or testing the quality of a part you'll need to run a restriction digest. We recommend using the following protocols.
 
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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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*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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=Overview=
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When using parts for 3A Assembly, or testing the quality of a part you'll need to run a restriction digest. We recommend using the following protocols.
  
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=Restriction Digest Protocol=  
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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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''estimated time: 30 min. active, 50 min. incubation''
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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]].
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=Restriction Digest Protocol=
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==Before You Start==
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''Estimated time: 30 min. active, 50 min. incubation''
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*You should keep all materials on ice.
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*At iGEM HQ we use restriction enzymes from New England Biolabs
  
 
==Materials==
 
==Materials==
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*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
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*Thermal cycler
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'''Notes:'''
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*You should keep all materials on ice.
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*At iGEM HQ we use restriction enzymes from New England Biolabs
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==Equipment==
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*Ice and bucket/container
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*Thermal cycler or heating block
  
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==Procedure==
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==Method==
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#Keep all enzymes and buffers used on ice.
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#Thaw NEB Buffer 2 and BSA in room temperature water. Mix by shaking the tubes, and flick/spin them to collect the liquid at the bottom of the tube.
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#Add 250ng of DNA to the appropriately labelled tube. Add distilled water to the tubes for a total volume of 16ul in each tube.
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Calculation example (with 25ng/ul as DNA sample concentration):
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250ng ÷ 25ng/ul = 10ul of DNA sample
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16ul (total volume) – 10ul (DNA sample) = 6ul of distilled water
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#Pipet 2.5ul of NEB Buffer 2 to each tube.
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#Pipet 0.5ul of BSA to each tube.
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#In the '''Part A''' tube: Add 0.5ul of EcoRI, and 0.5ul of SpeI.
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#In the '''Part B''' tube: Add 0.5ul of XbaI, and 0.5ul of PstI.
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#In the '''pSB1C3''' tube: Add 0.5ul of EcoRI, 0.5ul of PstI, and 0.5ul of Dpn1.
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#The total volume in each tube should be approximately 20ul. Mix well by pipetting slowly up and down. Spin the samples briefly to collect all of the mixture to the bottom of the tube.
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#Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes. We use a thermal cycler with a heated lid.
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#(''Optional, but recommended'') Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.
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#Use ~2ul of the digest (25ng of DNA) for ligations.
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<table class="aliquot">
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<tr class="aliquotheader"><td></td><td>Part A</td><td>Part B</td><td>linearized plasmid backbone</td></tr>
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<tr><th>DNA</th><td>250ng</td><td>250ng</td><td>250ng (10ul @ 25ng/ul)</td></tr>
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<tr><th>dH2O</th><td>adjust to 16ul</td><td>adjust to 16ul</td><td>6ul</td></tr>
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<tr><th>NEB Buffer 2</th><td>2.5ul</td><td>2.5ul</td><td>2.5ul</td></tr>
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<tr><th>BSA</th><td>0.5ul</td><td>0.5ul</td><td>0.5ul</td></tr>
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<tr><th>Enzyme 1</th><td>0.5ul EcoRI</td><td>0.5ul XbaI</td><td>0.5ul EcoRI</td></tr>
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<tr><th>Enzyme 2</th><td>0.5ul SpeI</td><td>0.5ul PstI</td><td>0.5ul Pst1</td></tr>
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<tr><th>Enzyme 3</th><td> </td><td> </td><td>0.5ul DpnI</td></tr>
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==Single Reaction==
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#Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul.<br>
 
#Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul.<br>
 
#Add 2.5ul of NEBuffer 2.<br>
 
#Add 2.5ul of NEBuffer 2.<br>
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#Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.  
 
#Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.  
  
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=Other Resources=
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==Video==
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<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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*''Please note, this video may be outdated.''
  
 
{{HelpPage/Footer}}
 
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Latest revision as of 15:49, 20 June 2017

Overview

When using parts for 3A Assembly, or testing the quality of a part you'll need to run a restriction digest. We recommend using the following protocols.

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 Protocol

Before You Start

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

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

Materials

  • (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

Equipment

  • Ice and bucket/container
  • Thermal cycler or heating block

Method

  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.


Other Resources

Video

Restriction Digest from iGEM Videos.

  • Please note, this video may be outdated.