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interactome:experiments:20191129 [2019/11/29 12:49] dan |
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| {{tag>protocol POEM N-termDHFR interactome}} | {{tag>protocol POEM N-termDHFR interactome}} | ||
| - | ## Back bone/ insert preparation for generating pDN0511 | + | ## RE check of potential pDN0513 |
| #### Material | #### Material | ||
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| | | Purified PCR product | | | POEM5 & POEM6 | | | | | Purified PCR product | | | POEM5 & POEM6 | | | ||
| | | ddH2O | Invurtogen | 10977-015 | | | | | | ddH2O | Invurtogen | 10977-015 | | | | ||
| - | | | I-CeuI | NEB | | | 37C, Inactivated at 80C | | + | | | I-XhoI | NEB | | | 37C, Inactivated at 80C | |
| - | | | I-SceI | NEB | | | 37C, Inactivated at 65C | | + | | | I-XbaI | NEB | | | 37C, Inactivated at 65C | |
| | | 10x Cutsmart buffer | NEB | B7204 | | | | | | 10x Cutsmart buffer | NEB | B7204 | | | | ||
| | | FasGene PCR clean kit | Nippon Genetics | FG-91302 | | | | | | FasGene PCR clean kit | Nippon Genetics | FG-91302 | | | | ||
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| ^ Check ^ Reagent ^ Volume (µl) per reaction ^ Final concenration |^ Memo ^ | ^ Check ^ Reagent ^ Volume (µl) per reaction ^ Final concenration |^ Memo ^ | ||
| - | | | 10x CS buffer | 3 | 1 | x | | | + | | | 10x CS buffer | 5 | 1 | x | | |
| - | | | DNA | 21 | | | For gel extraction | | + | | | DNA | 2 | | | For gel extraction | |
| - | | | I-CeuI | 3 | | | | | + | | | XbaI | 1 | | | | |
| - | | | I-SceI | 3 | | | | | + | | | XhoI | 1 | | | | |
| - | | ^ Total (µL) ^ 30 | | | | | + | | ^ Total (µL) ^ 10 | | | | |
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