Isothermal mix:
5x isothermal reaction buffer recipe:
| Reagent | Need | Notes |
|---|---|---|
| 1M Tris HCl pH 7.5 | 3mL | |
| 1M MgCl2 | 300µL | |
| 10mM dNTP mix | 600µL | thermofisher cat # 18427088; $555 per 1 mL |
| 1M DTT | 300µL | |
| Peg-8000 | 1.8g | |
| NAD | 20mg | |
| ddH2O | up to 6mL |
To make working concentration from 5X buffer:
| Reagent | Need | Notes |
|---|---|---|
| 1 aliquot of 5x buffer | 320µL | |
| T5 exonuclease | 1.2µL | ThermoFisher Cat #M0663S NEB; $69 / 1mL |
| Phusion Polymerase | 20µL | ThermoFisher cat #F530S; $121 per 10 µL |
| Taq ligase (M0208L) | 160µL | NEB Cat # M0208L; $332 per 250 µL |
| ddH2O | 700µL |
Aliquot into 15µL reactions in PCR tubes and store in -20˚C.
(Makes 2.4mL aka 160 aliquots of 15 µL)