Oct 21, 2021
Cleavage assay
This protocol is a draft, published without a DOI.
- 1iGEM Gifu
- iGEM Gifu
Protocol Citation: Yuichiroh Ikagawa 2021. Cleavage assay. protocols.io https://protocols.io/view/cleavage-assay-bzdmp246
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: In development
We are still developing and optimizing this protocol
Created: October 21, 2021
Last Modified: October 21, 2021
Protocol Integer ID: 54413
Abstract
Cleavage assay to validate for Cas12a activity
Materials
Reagents
Cas12a
crRNA
target DNA (U94)
reaction buffer ( PBS (pH 7.5), 150 mM KCl, 10 mM MgCl2, 1% glycerol and 0.5 mM DTT)
Thawed crRNA solution on ice.
Add the following solution to the tube and gently mix by pipetting.
| A | B | |
| COMPONENT | VOLUME (µl) | |
| Cas12a | 4 | |
| crRNA | 5 | |
| reaction buffer | 7 | |
| Total Volume | 16 |
If the crRNA or Cas12a solution was not added to the reaction as a negative control, the reaction buffer for it was added and the total volume was adjusted to 20 μl.
Incubate for 10 minutes.
Add 4μl of U94 solution to each tube.
Incubate the mixture at 37°C overnight.
Check for cleavage by1.5% agarose gel electrophoresis.