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| PCR Product | DNA Concentration | DNA volume | DNA weight |
|---|---|---|---|
| mKate | 26.7 ng/uL | 14 uL | 373.8 ng = 0.3738 ug |
| eYFP | 47.7 ng/uL | 14 uL | 667.8 ng = 0.6678 ug |
Typical restriction digest protocol: https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions
| Chemical | Quantity |
| Restriction Enzyme | 10 units is sufficient, generally 1µl is used |
| DNA | 1 µg |
| 10X NEBuffer | 5 µl (1X) |
| Total Reaction Volume | 50 µl |
| Incubation Time | 1 hour* |
| Incubation Temperature | Enzyme dependent |
For DpnI digest:
What do we use as the DNA concentration for the template DNA? Started at 1ng/uL in initial reaction.
NEBuffer 2.1 was selected for best performance in subsequent Golden Gate. It is very similar to the T4 ligase buffer used in the Golden Gate reaction.
Conditions for DpnI digest:
| Chemical | mKate | eYFP |
|---|---|---|
| Starting PCR reaction | 29 uL | 24 uL |
| DpnI | 2 uL | 1.7 uL |
| NEBuffer 2.1 | 4 uL | 3.4 uL |
| dH2O | 5 uL | 5.9 uL |
| TOTAL | 40 uL | 35 uL |
Incubated at 37C for 30 minutes. Heat shock at 80C for 20 minutes.
Total reaction volume - don't want to dilute too much?