Personal Interactome project | Proof of Concept Y2H/DHFR-PCA with HEK293T
pENTRY insert check
Amplifying insert region using ORFeome_Seq_Frd/Rvs.
Protocol
1 Reaction preparation
List of paramater tested.
| Tm/Target gene | MORF4L1 | MRFAP1 | BCL2L2 | BIK | MRFAP1L1 | |
|---|---|---|---|---|---|---|
| Row/Col on PCR plate | 1 | 3 | 4 | 5 | 6 | |
- Mix the following reagents in one tube (ideally from top to bottom)
| Check | Reagent | Volume (µl) 1 reaction | Volume (µl) for 50 reactions with 20µL vol | Final concenration | Memo | |
|---|---|---|---|---|---|---|
| ddH20 | 5.12 | 512 | - | |||
| Phusion HF buffer (5x) | 2.0 | 200 | - | |||
| 25mM dNTPs | 0.08 | 8 | 0.2 | mM | Equal volume mix of 100mM dA/T/G/CTPs will give this solution. | |
| Phusion pol | 0.2 | 20 | - | Home brew Phusion pol | ||
| 100µM Frd Primer | 0.05 | 5 | 0.5 | µM | ||
| 100µM Rvs Primer | 0.05 | 5 | 0.5 | µM | ||
| Aliquot 15 µL of master mix to each well. | ||||||
| Add 5 µL of 1/20 diluted bacterial culture to aliquoted master mix. | ||||||
| Total (µL) | 10 | 20 | ||||
2 Reaction
- Run the following program on a thermal cycler
| STEP | Action | Time | Memo |
|---|---|---|---|
| 1 | 98˚C | 10 min | Initial denaturation. |
| 2 | 98˚C | 30 sec | Denaturation. |
| 3 | 61˚C | 30 sec | Annealing. |
| 4 | 72˚C | 1 min | Extension. 15sec/kbp, but using longer |
| 5 | GOTO 2 | x 29 | |
| 6 | 72˚C | 5 min | Final extension. |
| 7 | 4˚C | FOREVER | Stop reaction. |
— Dan Yamamoto-Evans 2019/11/25 14:48
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