Personal Interactome project
Proof of Concept Y2H/DHFR-PCA with HEK293T
Generating template for barcoded cDNA generation primer from barcoded library samples.
Protocol
1 Reaction preparation
List of paramater tested.
- 10 different barcodes (purified plasmid sample) x 2 barcode types
- MORF4L1-40bp overlap for all genes
- 5 pilot genes with 2 barcodes each.
- Frd primer : BCamplif-Frd-MORF4L1-v2-4;PI-34-BClib-reamp-MRFAP1-Frd;PI-35-BClib-reamp-BCL2L2-Frd;PI-36-BClib-reamp-BIK-Frd;PI-37-BClib-reamp-MRFAP1L1-Frd
- Rvs primer : PI-33-Barcode-reamp1-Rvs
- 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 | 6.12 | 306 | - | |||
| 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. | |
| DNA tempalte (10ng/µL) | 1 | 50 | Diluted plasmid DNA sample. | |||
| Transfer the tube on ice | ||||||
| Phusion pol | 0.2 | 10 | - | Home brew Phusion pol | ||
| Aliquot 17 µL to each tube | ||||||
| 10µM Frd Primer | 0.5 | 1 | 0.5 | µM | ||
| 10µM Rvs Primer | 0.5 | 1 | 0.5 | µM | ||
| Fusion PCR template | 0.5 | 1 | ||||
| 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 | 30 sec | Denaturation. |
| 3 | 65 ˚C | 30 sec | Annealing. See Tm table |
| 4 | 72˚C | 30 sec | 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
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