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.
 
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=Overview=
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At iGEM HQ we recommend using the following protocols. The large reaction protocol will have enough of a volume for ligation and you'll be able to use a portion to run a gel. The small reaction protocol will only have enough volume for
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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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==Materials==  
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*PCR tube
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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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*dH20
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*Enzymes (EcoRI, XbaI, SpeI, PstI)
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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
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==Materials==
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*(1) 8-tube strip, or (3) 0.6ml thin-walled tubes 
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*BioBrick Part in BioBrick plasmid (Purified DNA, > 16ng/ul)
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*dH2O
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*NEB Buffer 2
 
*BSA
 
*BSA
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*Enzyme Buffer (NEBuffer 2)*
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*Restriction Enzymes: EcoRI, SpeI, XbaI, PstI
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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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==Method==
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#Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul.<br>
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#Add 2.5ul of NEBuffer 2.<br>
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#Add 0.5ul of BSA.<br>
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#Add 0.5ul of EcoRI.<br>
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#Add 0.5ul of PstI.<br>
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#There should be a total volume of 20ul. Mix well and spin down briefly.<br>
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#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''
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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.
  
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Notes: You should keep all materials on ice.
 
  
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==Protocol==
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=Other Resources=
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1. Add 500ng of DNA to be digested, and adjust with dH20 for a total volume of 42.5ul.<br>
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2. Add 5ul of NEBuffer 2 to the tube.<br>
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3. Add 0.5ul of BSA to the tube.<br>
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4. Add 1ul of your first enzyme.<br>
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5. Add 1ul of your second enzyme.<br>
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6. There should be a total volume of 50ul. Mix well and spin down.<br>
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7. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes.<br> ''We incubate in a thermocycler with a heated lid''<br>
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8. Run a portion of the digest on a gel, to check that both plasmid and part length are accurate. You may also use 2ul of the digest (20ng of DNA) for ligations.
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==Video==
  
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[[Linearized Plasmid Backbones Original Protocol]]
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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.''
  
 
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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.