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Restriction Enzyme Activity drop down quickly at 37C, so, always keep enzyme on ice when not in reaction
Materials
- DNA to be analysed
- Restriction enzyme(s) kit (Promega Cat#)
- ThermoMixer R
- Pipette and aerosol tips
- Centrifuge
- DNA, RNase-free 1.5ml tubes
- Ice
- Agarose gel reagents and apparatus
- UV light lamp (or Typhoon Imager)
Procedure
- Set up reaction mixture on ice as in the table below:
| Component |
quantity |
| H2O |
Scale up to 20 ul |
| 10X Restriction Buffer |
2 ul |
| DNA |
0.5~1 ug |
| Restriction Enzyme |
10 units |
- Mix, Spin. Incubate at 37C for 1 hr.
- Take 5ul reaction + 4ul H2O + 1ul loading buffer, mix.
- Heat at 56C for 2 min then cool on ice immediately.
- Spin. Load on to agarose gel and run at 80V, check under UV light or scan on Typhoon Imager. Estimate fragments size by comparing with DNA ladder.
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