Restriction Digest: Difference between revisions
Jump to navigation
Jump to search
(Created page with " Protocol 1. Check the NEB (or other supplier) website to confirm enzyme compatibility 2. In a PCR tube add the reaction components according to the table listed below 3. Incubate at 37°C for 30 min if time-saver qualified, 60 minutes if not Example Reaction Component Volume ddH2O Up to 50 µL 10X rCutSmart Buffer 5 µL DNA x (1 µg) Enzyme 1 1 µL Enzyme 2 1 µL Notes - NEB High-Fidelity (HF) enzymes are ordered whenever possible, they all use rCutSmart reactio...") |
No edit summary |
||
| Line 1: | Line 1: | ||
Protocol | '''Protocol''' | ||
# Check the NEB (or other supplier) website to confirm enzyme compatibility | |||
# In a PCR tube add the reaction components according to the table listed below | |||
# Mix reaction gently and spin down briefly with benchtop centrifuge | |||
# Incubate at 37°C for 30 min if time-saver qualified, 60 minutes if not | |||
Notes | '''Example Reaction''' | ||
- NEB High-Fidelity (HF) enzymes are ordered whenever possible, they all use rCutSmart reaction buffer and are usually time-saver qualified | {| class="wikitable" | ||
- NEB has the option to select your specific enzymes and generate a detailed protocol | |'''Component''' | ||
|'''Volume''' | |||
|- | |||
|ddH2O | |||
|Up to 50 µL | |||
|- | |||
|10X rCutSmart Buffer | |||
|5 µL | |||
|- | |||
|DNA | |||
|x (1 µg) | |||
|- | |||
|Enzyme 1 | |||
|1 µL | |||
|- | |||
|Enzyme 2 | |||
|1 µL | |||
|} | |||
'''Notes''' | |||
- NEB High-Fidelity (HF) enzymes are ordered whenever possible, they all use rCutSmart reaction buffer and are usually time-saver qualified | |||
- NEB has the option to select your specific enzymes and generate a detailed protocol | |||
- Heat inactivation may be recommended if there are additional steps in the workflow without a DNA purification step | - Heat inactivation may be recommended if there are additional steps in the workflow without a DNA purification step | ||
References, related resources, and acknowledgments | |||
'''References, related resources, and acknowledgments''' | |||
https://nebcloner.neb.com/#!/redigest | |||
Latest revision as of 18:56, 17 January 2025
Protocol
- Check the NEB (or other supplier) website to confirm enzyme compatibility
- In a PCR tube add the reaction components according to the table listed below
- Mix reaction gently and spin down briefly with benchtop centrifuge
- Incubate at 37°C for 30 min if time-saver qualified, 60 minutes if not
Example Reaction
| Component | Volume |
| ddH2O | Up to 50 µL |
| 10X rCutSmart Buffer | 5 µL |
| DNA | x (1 µg) |
| Enzyme 1 | 1 µL |
| Enzyme 2 | 1 µL |
Notes
- NEB High-Fidelity (HF) enzymes are ordered whenever possible, they all use rCutSmart reaction buffer and are usually time-saver qualified
- NEB has the option to select your specific enzymes and generate a detailed protocol - Heat inactivation may be recommended if there are additional steps in the workflow without a DNA purification step
References, related resources, and acknowledgments