POEM project | N-term DHFR-PCA plasmid construction
Overview
Step | Description | Date/Initials | Link protocol | Link results |
---|---|---|---|---|
1 | Digest pDN0501 / pDN0502 with HidIII-HF and NotI-HF | 20191118KM | ||
2 | Amplify DHFR-attL1 fragments ordered by TWIST | 20191122DEY | ||
3 | Digest amplified DHFR-attL1 fragments with HidIII-HF and NotI-HF | 20191123DEY | ||
4 | T4 ligation | |||
5 | Transformation to ccdB survival cells |
Material
Check | Reagent | Supplier | Cat. no | Primer/Sample ID | Memo |
---|---|---|---|---|---|
Purified PCR product | POEM5 & POEM6 | ||||
ddH2O | Invurtogen | 10977-015 | |||
HindIII-HF | NEB | R3104S | 37C, Inactivated at 80C | ||
NotI-HF | NEB | R3189S | 37C, Inactivated at 65C | ||
10x Cutsmart buffer | NEB | B7204 | |||
FasGene PCR clean kit | Nippon Genetics | FG-91302 |
Protocol
1 Reaction preparation
- Mix the following reagents in one tube (ideally from top to bottom)
Check | Reagent | Volume (µl) per reaction | Final concenration | Memo | |
---|---|---|---|---|---|
ddH20 | 34 | - | |||
10x CS buffer | 5 | 1 | x | ||
DNA | 10 | 1 µg should be enough | |||
NotI-HF | 1 | 20U/µL | |||
HindII-HF | 1 | 20U/µL | |||
Total (µL) | 50 |
2 Reaction
- Run the following program on a thermal cycler
STEP | Action | Time | Memo |
---|---|---|---|
1 | 37˚C | 16 hours | Reaction |
2 | 80˚C | 20 mins | Inativation |
7 | 4˚C | FOREVER | Stop reaction. |
3 Cleanup purification
- Purify product using FastGene PCR clean kit.
- Measure concentration
— Dan Yamamoto-Evans 2019/11/23 12:15
Print at 96%