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POEM project | N-term DHFR-PCA plasmid construction
RE check of potential pDN0513 samples.
Material
| Check | Reagent | Supplier | Cat. no | Primer/Sample ID | Memo |
|---|---|---|---|---|---|
| Purified PCR product | POEM5 & POEM6 | ||||
| ddH2O | Invurtogen | 10977-015 | |||
| XbaI | NEB | 37C, Inactivated at 80C | |||
| XhoI | 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 | |
|---|---|---|---|---|---|
| ddH20 | 5 | - | |||
| 10x CS buffer | 1 | 1 | x | ||
| DNA | 2 | 1 µg should be enough | |||
| NotI-HF | 1 | 20U/µL | |||
| HindII-HF | 1 | 20U/µL | |||
| Total (µL) | 10 | ||||
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. |
Sanger sequence sample with right band pattern and proceed to I-SceI/I-CeuI digestion.
— Dan Yamamoto-Evans 2019/11/29 12:33
Print at 96%