Oct 24, 2020
Restriction Digest
- Zhujun Wei1
- 12020 iGEM NEFU China
- 2020 iGEM NEFU China
Protocol Citation: Zhujun Wei 2020. Restriction Digest. protocols.io https://dx.doi.org/10.17504/protocols.io.bnwvmfe6
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: October 24, 2020
Last Modified: October 24, 2020
Protocol Integer ID: 43701
Keywords: restriction digest this protocol, restriction digest, different methods of digestion, digestion, different method, protocol
Abstract
This protocol includes three different methods of digestion.
Materials
Restriction enzyme
Buffer 2.1/3.1/CutSmart
Safety warnings
Please wear gloves for the experiment.
Add the following reagents to a tube.
| Total | 10ul | 30ul | 50ul | |
| DNA | 0.4ug | 1.2ug | 2ug | |
| Enzyme | 0.2ul | 0.6ul | 1ul | |
| Buffer(10*) | 1ul | 3ul | 5ul | |
| ddwater Fill the rest with water |
Pipette up and down thoroughly.
Put at 37℃ for about 2 hours (according to different enzymes).
Add the following reagents to a tube.
| Total | 10ul | 30ul | 50ul | |
| DNA | 0.4ug | 1.2ug | 2ug | |
| Enzyme | 0.2ul | 0.6ul | 1ul | |
| Buffer(10*) | 1ul | 3ul | 5ul | |
| ddWater | Fill the rest with water | Fill the rest with water | Fill the rest with water |
Pipette up and down thoroughly.
Put at 37℃ for about 2 hours (according to different enzymes).
Put at 65℃ for about 20 min.(according to different enzymes)
Purification of enzyme digestion products with Cycle Pure Kit D6492（OMEGA bio-tek）
DNA product concentrations were measured using Nano
Add the following reagents to a tube:
| Total | 10ul | 30ul | 50ul | |
| DNA product | 0.4ug | 1.2ug | 2ug | |
| CIP(NEB) | 0.2ul | 0.6ul | 1ul | |
| Cutsmart(10*) | 1ul | 3ul | 5ul | |
| ddWater | Fill the rest with water | Fill the rest with water | Fill the rest with water |
Pipette up and down thoroughly.
Put at 37℃ for about 2 hours (according to different enzymes).
Add the following reagents to a tube.
| Total | 10ul | 30ul | 50ul | |
| DNA | 0.4ug | 1.2ug | 2ug | |
| Enzyme 1 | 0.2ul | 0.6ul | 1ul | |
| Enzyme 2 | 0.2ul | 0.6ul | 1ul | |
| Buffer(10*) | 1ul | 3ul | 5ul | |
| ddwater | Fill the rest with water | Fill the rest with water | Fill the rest with water |
Pipette up and down thoroughly.
Put at 37℃ for about 2 hours (according to different enzymes).
Add the following reagents to a tube.
| Total | 10ul | 30ul | 50ul | |
| DNA product | 0.4ug | 1.2ug | 2ug | |
| DpnI(NEB) | 0.2ul | 0.6ul | 1ul | |
| Cutsmart(10*) | 1ul | 3ul | 5ul | |
| ddwater | Fill the rest with water | Fill the rest with water | Fill the rest with water |
Pipette up and down thoroughly.
Put at 37℃ for about 2 hours (according to different enzymes).