Feb 02, 2025

Public workspaceVector Digestion and Purification V.3

DOI
dx.doi.org/10.17504/protocols.io.261ge5dyog47/v3
  • 1Washington University
Cecilia Escudero
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DOI: dx.doi.org/10.17504/protocols.io.261ge5dyog47/v3
Protocol Citation: Carolina Lopez 2025. Vector Digestion and Purification. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5dyog47/v3Version created by Carolina Lopez
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: May 28, 2024
Last Modified: February 02, 2025
Protocol Integer ID: 118321
Abstract
Protocol for plasmid digestion and purification
Materials
Reagents:
  • Restriction Enzymes (New England Biolabs)
  • 10x Cutsmart Buffer (New England Biolabs)
  • Agarose
  • EtBr
  • NEB Monarch DNA Gel Extraction kit (NEB #T1020)
Digestion:
Digestion:
1h 10m
1h 10m
1. Double digest Vector with New England Biolabs Restriction Enzymes: 80uL reaction

AB
ComponentVolume (uL)
DNA plasmidX ul for 4 ug of plasmid
10x Cutsmart Buffer8 uL
Enzyme 14 uL
Enzyme 24 uL
NucleaseFree H2O64-X uL
2. Incubate for Duration00:30:00 at Temperature37 °C .
3. Add Amount16 µL of 6x loading Dye to reaction and vortex briefly to mix.
4. Make 1% agarose gel.
a) Mix 1 g of Agar with Amount100 mL of TAE Buffer.
b) Microwave to boil agarose and let cool until you can touch bottle, but gel is not solid.
c) Add Amount2 µL of EtBr to agarose and pour into DNA gel mold with 10 well comb.
d) Let gel solidify.
5. Load Amount100 µL of reaction into well of gel
6. Run gel for Duration00:40:00 at 120V.
7. Visualize band with UV light and cut out section of gel with band with new razor blade and place in Amount1.5 mL tube.

Notes:
  • Not all two enzymes can be used together in a double digestion. Think about it ahead and you can test your two enzymes here: https://nebcloner.neb.com/#!/redigest.
  • Enzymes are different, but most can be digested in CutSmart buffer at 37°C within 30 minutes. This is a general protocol. Please check the best digestion conditions for your enzymes in this web：https://nebcloner.neb.com/#!/redigest. It will provide the temperature, buffer, and incubation time specific to the two enzymes you are using.
1h 10m
Purification bands from gel with Monarch DNA Gel Extraction kit:
Purification bands from gel with Monarch DNA Gel Extraction kit:
  1. Weigh the gel slice.
  2. Add 4 volumes of Monarch Gel Dissolving Buffer to the tube with the gel slice (e.g., 400 μl buffer per 100 mg agarose). If the gel slice is >150 mg, consider reducing the amount of Gel Dissolving Buffer to 3 or 3.5 volumes to minimize the guanidine salt present in the workflow.
  3. Incubate the sample between 37–55°C (typically 50°C), inverting periodically until the gel slice is completely dissolved (generally 5–10 minutes).
  4. Insert the column into collection tube and load sample onto the column. Spin for 1 minute, then discard flow-through.
  5. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
  6. Repeat wash (Step 5).
  7. Re-spin the column and tubes.
  8. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If in doubt, re-spin for 1 minute before placing into clean microfuge tube.
  9. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, and spin for 1 minute to elute DNA.

Notes:
  • Step2: If the volume of the dissolved sample exceeds 800 μl, the loading of the sample onto the column should be performed in multiple rounds to not exceed the volume constraints of the spin column.
  • Step3: For DNA fragments > 8 kb, an additional 1.5 volumes of water should be added after the slice is dissolved to mitigate the tighter binding of larger pieces of DNA (e.g., 100 mg gel slice: 400 μl Gel Dissolving Buffer: 150 μl water). Failure to dissolve all the agarose will decrease the recovery yield due to incomplete extraction of the DNA and potential clogging of the column by particles of agarose.
Protocol references