DpnI Digest
Ran DpnI restriction digest on products from PCRs of mKate and eYFP. The starting plasmids in this case (pENTR_L1_mKate_L2 and pENTR_L1_EYFP_L2) both have markers for kanamycin resistance, which is the same selection marker we are using for our Golden Gates (since our GGDonr plasmid has a kanamycin resistance marker). The DpnI digest will allow us to separate out the template DNA (starting plasmid) from the PCR purification because DpnI only cuts methylated DNA and cuts it frequently (it has a 4bp recognition site). Thus, the template DNA will be degraded and the desired PCR product will remain intact.
| 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.
Total reaction volume - don't want to dilute too much?