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POEM project | N-term DHFR-PCA plasmid construction
Back bone/ insert preparation for generating pDN0511
Material
| Check | Reagent | Supplier | Cat. no | Primer/Sample ID | Memo |
|---|---|---|---|---|---|
| Purified PCR product | POEM5 & POEM6 | ||||
| ddH2O | Invurtogen | 10977-015 | |||
| I-CeuI | NEB | 37C, Inactivated at 80C | |||
| I-SceI | NEB | 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 | |
|---|---|---|---|---|---|
| 10x CS buffer | 3 | 1 | x | ||
| DNA | 21 | For gel extraction | |||
| I-CeuI | 3 | ||||
| I-SceI | 3 | ||||
| Total (µL) | 30 | ||||
2 Reaction
- Run the following program on a thermal cycler
| STEP | Action | Time | Memo |
|---|---|---|---|
| 1 | 37˚C | 1 hour | Reaction |
| 2 | 80˚C | 20 mins | Inativation |
| 7 | 4˚C | FOREVER | Stop reaction. |
Run gel and extract band.
— Dan Yamamoto-Evans 2019/11/29 12:33
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