Personal Interactome project
Proof of Concept Y2H/DHFR-PCA with HEK293T
Re-trying with additional cycle to amplify MORF4L1. The annealing temp is set to 52C.
Overview
| Step | Description | Date/Initials | Link protocol | Link results |
|---|---|---|---|---|
| 1 | Total RNA extraction from HEK293T cells | 20191122DEY | ||
| 2 | 1st strand cDNA synthesis | 20191122DEY | ||
| 3 | Target gene amplification from cDNA sample | Current | ||
| 4 | BP-reaction | |||
| 5 | Sanger sequence check |
Material
| Check | Reagent | Supplier | Cat. no | Primer/Sample ID | Memo |
|---|---|---|---|---|---|
| Nuclease free ddH2O | Invitrogen | 10977-015 | |||
| Phusion HF buffer (5x) | NEB | B0518S | |||
| 25mM dNTPs | TaKaRa | ||||
| Frd Primer | FASMAC | PI11 to PI20 | |||
| Rvs Primer | FASMAC | PI11 to PI20 | |||
| Phusion pol | - | KM2 | Home brew Phusion pol | ||
| Template cDNA | - | Prepared on 20191122. Sample1 | |||
| Agarose | RIKAKEN | RSV-AGRP-500G | |||
| 1x TBE buffer | - | - | Homebrew (link:) | ||
| ExcelBand 100 bp DNA Ladder | SMOBiO | DM2100 | |||
| 5x Loading dye | - | - | Homebrew (link:) | ||
| Midori green Advance | Nippon Genetics | NE-MG04 |
Before preparing: Check your primer Tm at NEB website (http://tmcalculator.neb.com/#!/main with Phusion/HF buffer setting)
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 | |
| 52.0 | A | ||||||||||
- 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 | 10 sec | Initial denaturation. |
| 2 | 98˚C | 10 sec | Denaturation. |
| 3 | 52˚C | 10 sec | Annealing. |
| 4 | 72˚C | 1 min | Extension. 15sec/kbp, but using longer |
| 5 | GOTO 2 | x 39 | |
| 6 | 72˚C | 10:00 | Final extension. |
| 7 | 4˚C | FOREVER | Stop reaction. |
3 Agarose gel electrophioresis
- Make agarose gel
| Reagent | Amount | Memo | |
|---|---|---|---|
| Agarose powder | 1.0 g | 1.0%(w/v) | |
| 1x TBE buffer | 100 mL | ||
| Microwave and dissolve agarose completely. | |||
| Midori green advance | 10 µL | DNA stain | |
| Swirl to mix the stain uniformly, and cast gels. 100 mL will give you 2 x wide + 2 x small on heat-tolerant trays. | |||
| Wait for gel to settle. | |||
— Dan Yamamoto-Evans 2019/11/25 14:48
Print at 96%