• Inoculate overnight culture in LB + antibiotic (TET for HT115(DE3) strain) (2-5ml). Shake overnight at 37C.

• Inoculate 25 ml LB + antibiotic with overnight culture, 1:100 dilution. Grow cells to OD595= 0.4. Can grow cells in 50 ml sterile centrifuge tube.

• Spin cells 10 min 3000 rpm at 4C.

• Resuspend pellet in 0.5X original volume cold, sterile 50 mM CaCl2 (12.5ml). Resuspend by GENTLY pipetting up and down a few times with a wide bore pipet--no vortexing.

• Incubate on ice 30 min.

• Spin as before at 4C.

• Resuspend pellet as before in 0.1X original volume CaCl2 (2.5ml). Keep cells cold (4C).

• Use 50-200 ul for transformation. Cells can be used as is for up to three days (stored at 4C).

• The cells can be frozen by: adding glycerol to final concentration of 10%, rapid freezing on dry ice/EtOH, and storing at –80.

Cloning Bootcamp

• 37C heatblock
• 42C heatblock
• Age1
• 10X buffer NEB1.1
• rSAP
• Sterile water for cloning
• Ligase + 2X rapid lligase buffer
• 1 sample: PCR cleanup kit (Qiaquick) (one per group)
• 1 tube (5 rxns) Competent cells (DH5 alpha, just for cloning)
• 5x LB+Amp plates
• Sterile beads for spreading
• 30 ng/ul backbone plasmid for cutting (estimated 15n/ul after elution w/ 30ul)
• Gel purified inserts (gamma tub, centrin, sas-6 at 15ng/ul)
• LB (plain) for transformation recovery

Naegleria 101 (2/7/22)

Activities:

• Differentiate cells,
• watch amoebae (phase microscopy),
• look at flagellates.
• Pick colonies for cloning.

Materials:

• 4 LB+ Amp +
• Tubes for growing cultures from previous lab’s cloning
• Toothpicks for above

8 QIAprep Spin miniprep kits for 4 cultures each

Notes before starting

• Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000.
• Add the provided RNase A solution to Buffer P1, mix and store at 2–8°C.
• Add ethanol (96–100%) to Buffer PE before use (see bottle label for volume).
• All centrifugation steps are carried out at 13,000 rpm (~17,900 x g) in a conventional table-top microcentrifuge.

Each kit contains:

• 4 spin columns
• 6 buffers, as below

Other materials

• Age1 + cutsmart
• 8 2mL tubes of LB broth
• competent HT115 cells from -80
• two 1% gels for wide gel rig, 2 small-well combs each
  • 2 g agarose
  • 200 mL 1X TAE
  • 20 uL SYBR Safe
• 1X TAE running buffer
• 1KB DNA standard

Equipment

• Microcentrifuges capable of 13000rpm
• Hot blocks at
  • 37
  • 42
• two wide gel rigs
• two power supplies
• two blue light boxes with photography box

M Feb 14

32 (?) tubes of LB-amp