Protocol for standard PCR
Material
- Sample : DNA template
- Primers
- dNTPs
- Phusion buffer
- Phusion pol
Before preparing: Check your primer Tm at NEB website (http://tmcalculator.neb.com/#!/main with Phusion/HF buffer setting)
Protocol
1 Reaction preparation
- Mix the following reagents in one tube (ideally from top to bottom)
| Check | Reagent | Volume (µl) 1 reaction | Volume (µl) _ _ _ reaction | Final concenration | Memo | |
|---|---|---|---|---|---|---|
| ddH20 | 6.62 | - | ||||
| Phusion HF buffer (5x) | 2.0 | - | ||||
| 25mM dNTPs | 0.08 | 0.2 | mM | Equal volume mix of 100mM dA/T/G/CTPs will give this solution. | ||
| Frd Primer | 0.05 | 0.5 | µM | |||
| Rvs Primer | 0.05 | 0.5 | µM | |||
| Transfer the tube on ice | ||||||
| Phusion pol | 0.2 | - | Home brew Phusion pol | |||
| Template | 1 | - | 1ng is enough for template. | |||
| Total (µL) | 10 | |||||
2 Reaction
- Run the following program on a thermal cycler
| STEP | Action | Time | Memo |
|---|---|---|---|
| 1 | 98˚C | 30 sec | Initial denaturation. |
| 2 | 98˚C | 10 sec | Denaturation. |
| 3 | 65˚C | 10 sec | Annealing. Depending on primer Tm |
| 4 | 72˚C | 15 sec | Extension. Depends on amplicon length. |
| 5 | GOTO 2 | x 34 | |
| 6 | 72˚C | 5:00 | Final extension. |
| 7 | 4˚C | FOREVER | Stop reaction. |
3 Agarose gel electrophioresis
- Make agarose gel
| Reagent | Amount | Memo |
|---|---|---|
| Agarose powder | 1.0 g | This is for 1.0%. For “1kbp> Expected >500bp”, use 1.5%. For “500bp>Expected”, use 2.0%. |
| 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. | ||