Aug 11, 2022

Public workspaceDiagnostic Restriction Digest

This protocol is a draft, published without a DOI.
  • 1University of Wisconsin - Stout
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Protocol Citation: Brian Teague 2022. Diagnostic Restriction Digest. protocols.io https://protocols.io/view/diagnostic-restriction-digest-ce58tg9w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 11, 2022
Last Modified: August 11, 2022
Protocol Integer ID: 68512
Abstract
In this restriction digest, you'll use an enzyme that cuts DNA to cut your miniprepped plasmid. This can give you some evidence as to whether your plasmid is what you expected or not.



You'll also use Benchling to predict the result of your digest -- that way, you can compare your prediction to your actual result.
Materials
  • Miniprep DNA
  • ReagentPvuII-HF - 5,000 unitsNew England BiolabsCatalog #R3151S
  • ReagentCutSmart® BufferNew England BiolabsCatalog #B7204S
  • Nuclease-free water
  • A 200 ul PCR tube

Safety warnings
None of the materials we're using today are hazardous.
HOWEVER, we are shedding nucleases -- enzymes that degrade DNA -- all the time. Wear lab coats and gloves to keep your samples nuclease-free.
Perform the diagnostic digest
Perform the diagnostic digest
1h
1h
For each miniprep, compute the volume that contains Amount1 µg of DNA.

In the PCR tube, mix:
  • The volume of DNA you computed in step 1, up to a maximum of Amount5 µL
  • Amount2 µL of CutSmart enzyme buffer
  • Amount2 µL of PvuII enzyme
  • Enough nuclease-free water for a total volume of Amount20 µL



Mix by flicking the tube gently, then spin down briefly.
Incubate your reaction for Duration01:00:00 at Temperature37 °C

1h
Simulate the diagnostic digest on Benchling
Simulate the diagnostic digest on Benchling
1h
1h
While your digest is running, you can use Benchling to simulate your digest. Begin by opening the "Cas9 Plasmid" document (or whichever document your instructor indicates). Then, click the "scissors" icon on the right to open the restriction digest panel.



Pull down the "Enzyme Lists" drop-down and choose "NEB". In the "Find Enzyme" box, type "PvuII" (that's the letters "Pvu" and two upper-case i's). The enzyme list should just show "PvuII" -- click it.



Note
Helpful hint: Once you have chosen a list, you can see where on the plasmid each of them cuts by closing the restriction digest panel and selecting the "PLASMID" tab at the top.



Click the blue RUN DIGEST button at the bottom. The new tab that opens tells you the sizes of the DNA fragments were you to digest this plasmid with those enzyme(s).



There's also a new tab called VIRTUAL DIGEST. Click it. Change Ladder option to "NEB 2-Log", which is the ladder we are using in the lab. Voila, a simulation of the gel you can expect to see when you run the digest for real.



After your ditest is complete, proceed to analyze your restriction digest using gel electrophoresis.

Note
When you prepare your samples for the gel, mix Amount5 µL of loading dye with your entire digest and load the whole thing on the gel. There's no need to keep any around for future use.