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 |
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.