## Personal Interactome project

## Proof of Concept Y2H/DHFR-PCA with HEK293T

##### Re-trying with new cDNA sample.

#### Material

#### Protocol

##### 1 Reaction preparation

List of paramater tested.

RT | - | + | - | + | - | + | - | + | - | + |

Tm/Target gene | MORF4L1 | MORF4L1 | MRFAP1 | MRFAP1 | BCL2L2 | BCL2L2 | BIK | BIK | MRFAP1L1 | MRFAP1L1 |
---|---|---|---|---|---|---|---|---|---|---|

Row/Col on PCR plate | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |

Frd primer | PI 11 | PI 11 | PI 13 | PI 13 | PI 15 | PI 15 | PI 17 | PI 17 | PI 19 | PI 19 |

Rvs Primer | PI 12 | PI 12 | PI 14 | PI 14 | PI 16 | PI 16 | PI 18 | PI 18 | PI 20 | PI 20 |

Tm | 56 | 56 | 58 | 58 | 65 | 65 | 56 | 56 | 57 | 57 |

- Mix the following reagents in one tube (ideally from top to bottom)

Check | Reagent | Volume (µl) 1 reaction | Volume (µl) for 25 reactions with 20µL vol | Final concenration | Memo | |
---|---|---|---|---|---|---|

ddH20 | 6.62 | 331 | - | |||

Phusion HF buffer (5x) | 2.0 | 100 | - | |||

25mM dNTPs | 0.08 | 4 | 0.2 | mM | Equal volume mix of 100mM dA/T/G/CTPs will give this solution. | |

cDNA template | 1 | 50 | Diluted cDNA sample. | |||

Transfer the tube on ice | ||||||

Phusion pol | 0.2 | 10 | - | Home brew Phusion pol | ||

Aliquot 18 µL to each tube | ||||||

10µM Frd Primer | 0.5 | 1 | 0.5 | µM | ||

10µM Rvs Primer | 0.5 | 1 | 0.5 | µM | ||

Total (µL) | 10 | 20 |

##### 2 Reaction

- Run the following program on a thermal cycler

STEP | Action | Time | Memo |
---|---|---|---|

1 | 98˚C | 3 min | Initial denaturation. |

2 | 98˚C | 10 sec | Denaturation. |

3 | X ˚C | 30 sec | Annealing. See Tm table |

4 | 72˚C | 3 min | Extension. 15sec/kbp, but using longer |

5 | GOTO 2 | x 29 | |

6 | 72˚C | 5:00 | Final extension. |

7 | 4˚C | FOREVER | Stop reaction. |

— *Dan Yamamoto-Evans 2019/12/10 11:37*
**Print at 96%**