Template:DNA Stability Test Protocol
1. Transform cells with plasmid containing Ptet and self-lysis device. Use transformation protocol via: Template:SBB-Protocols_Micro1.
2. Create 20ng/mL atc: atc is currently at 2mg/mL. Dilute 1uL of 2mg/mL atc in 1mL of NEB to get a concentration of 2ug/mL of atc (1,000X).
3. Spin down 200uL of transformed cells, remove media supernatant.
4. Resuspend in 200uL of NEB buffer. Spin down and remove supernatant.
5. Resuspend & induce cell lysis with 200uL of 1,000X atc dilution.
6. Let incubate 30 minutes at 37C.
7. Digest 12uL plasmid DNA with 0.5uL AlwN1 enzyme and 1uL NEB buffer. Incubate at 37C for an hour.
8. Zymo gel purify digested plasmid.
9. Remove 20uL of lysate as a negative control that receives no digested plasmid.
10. Prep 8 tubes with 2uL of 100mM EDTA, labelled as timepoints 0, 5, 10, 15, 20, 30, 45 and 60.
11. Add 12uL (all) of predigested plasmid DNA (B10sbb23) to the lysate tube. Mix well. Immediately remove 20uL of lysate into the prepared timepoint 0 tube. Incubate remainder at 37C.
12. Inactivate nucleases at given time-points (0, 5, 10, 15, 20, 30, 45, 60) by adding 20uL of lysate to each timepoint tube containing EDTA and setting on ice.
13. Run gels to determine integrity of plasmid, with 1uL loading dye and 9uL lysate.