Oct 04, 2024
Version 2
Vector Digestion and Purification V.2
- 1Washington University
Protocol Citation: Carolina Lopez 2024. Vector Digestion and Purification. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5dyog47/v2Version 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: October 04, 2024
Protocol Integer ID: 109145
Abstract
Protocol for plasmid digestion and purification
Materials
Reagents:
- Restriction Enzymes (New England Biolabs)
- 10x Cutsmart Buffer (New England Biolabs)
- Agarose
- EtBr
- Promega Wizard SV Gel and PCR Purification Kit (A9282)
Isolate Digested Vector:
Isolate Digested Vector:
3h 2m
3h 2m
1. Digest Vector with New England Biolabs Restriction Enzymes:
2. Incubate for 02:00:00 at 37 °C .
3. Add 10 µL of 6x loading buffer to reaction and vortex briefly to mix.
4. Make 1% low melt-agarose gel.
a) Mix 1 g of Agar with 100 mL of TAE Buffer.
b) Microwave to boil agarose and let cool until you can touch bottle, but gel is not solid.
c) Add 1.5 µL of EtBr to agarose and pour into DNA gel mold with 10 well comb.
d) Let gel solidify.
5. Load 60 µL of reaction into well of gel
6. Run gel for 00:45:00 at 120V.
7. Visualize band with UV light and cut out section of gel with band with new razor blade and place in 1.5 mL tube.
8. Purify Band from gel with Promega Wizard SV Gel and PCR Purification Kit (A9282)
b) Weigh DNA gel fragment and add 10 µL of Membrane Binding Solution per 10 mg of gel slice.
c) Incubate mixture at 65 °C for 00:10:00 or until gel is completely melted.
d) Add melted gel mixture to SV minicolumn in Collection Tube and incubate at room temperature for 00:01:00 .
e) Centrifuge at max speed for 00:01:00 . Discard flowthrough and reinsert column into tube.
f) Add 700 µL of Membrane Wash Solution. Centrifuge at max speed for 00:01:00 . Discard flowthrough and reinsert column into tube.
g) Add 500 µL of membrane wash solution. Centrifuge at max speed for 00:01:00 . Discard flowthrough and reinsert column into tube.
h) Spin empty column for 00:01:00 at max speed to remove excess ethanol.
i) Transfer column to labelled 1.5 mL tube and add 35 µL of NF H20. Incubate for 00:01:00 at room temperature.
j) Centrifuge at max speed for 00:01:00 . Keep eluate and store at -20 °C .
3h 2m