Team:UCLA/Notebook/Recombinant Expression/17 August 2015

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Solutions to Prepare for Rest of Summer

Solutions needed for gene expression, cell lysis, and protein purification of all recombinant silk products and their derivatives. Prepared by Jessica today.

Gene expression (store all at -20 deg)

• 1000x Chloramphenicol Stock Solution (34 mg/mL)
  • For 10 mL stock:
    • Dissolve 0.340g chloramphenicol solute in 10 mL of 100% EtOH in a fume hood in a conical tube.
    • Cap and vortex and until chloramphenicol goes into solution and dissolves.
    • Filter sterilize using a 0.22 um-sterile filter and 10mL syringe.
    • Store at -20 (no need to aliquot, we will use a lot of it).
  • Use at a final concentration of 1x (34 ug/mL) in a 1:1000 dilution of the final medium for use.

• 1000x Carbenicillin Stock Solution (50 mg/mL)
  • For 10 mL stock:
    • Dissolve 0.5g of carbenicillin salt in 9.5 mL of ddH2O (ultrapure).
    • Once dissolve, top up the volume to 10 mL total.
    • Filter sterilize using a 0.22 um-sterile filter and 10 mL syringe.
    • Store at -20 in 1mL aliquots.
  • Use at a final concentration of 1x (50ug/mL) in a 1:1000 dilution of the final medium for use.

• 100 mM IPTG stock solution
  • For 50 mL stock
    • Dissolve 1.19g of IPTG stock in 50 mL of ddH20 (ultra pure) in a 50 mL conical.
    • Filter sterilize using a 0.22um sterile filter and 60 mL syringe.
    • Aliquot into 1mL samples and store at -20.

IMAC Purification (Store all of these at 4 degrees Celsius

8x Binding Buffer: 40mM Imidazole, 4 M NaCl, 160 mM Tris-HCl (pH 8)

• For 50mL:
  • Add 0.13616g of Imidazole, 11.688 g NaCl, 8mL of Tris-Hcl (pH 8). Fill to the 50mL mark with DI H20.

• Use as a 10mL 1x binding buffer (1.875 mL 8x binding buffer, fill to the 15mL mark with DI water). Use as is (8x) as a wash buffer (40mM), and as a 15 mL 4x for a 20mM imidazole binding buffer (7.5 mL binding buffer with 7.5 mL H2O).

8x Elution Buffer Stock: 640 mM Imidazole, 4 M NaCl, 160 mM Tris-HCl (pH 8)

• For 50mL:
  • Add 11.688 g NaCl, 2.17g Imidazole, and 8mL of Tris-HCl (pH 8). Fill to the 50mL mark with DI H2O.

• To prepare a 100mM Imidazole stock, prepare a 15mL 1.25x dilution (2.34mL of elution buffer to 15 mL conical, and fill to mark). For 250mM Imidazole, prepare a 15mL 3.125x dilution (add 5.85mL of Elution buffer to 15 mL conical, and fill to mark). For 50mM Imidazole use 1.17 mL of 8x elution buffer to 15mL conical and fill to mark.

4x Strip Buffer: 400mM EDTA, 2 M NaCl, 80 mM Tris-HCl (pH 8)

• For 50mL:
  • Add 5.844g NaCl, 4 mL of tris-HCl, 40mL of EDTA. Fill to the mark with DI H20.
• To prepare 1x Strip Buffer, add 3.75mL 4x strip to 15 mL conical and fill to the top.

Picking colonies of M2 9/12/15 mer for regrowth

Prepared three tubes with 10 mL LB and supplemented with 10 uL Chlor. Picked one colony off each plate, and inoculated in the broth, swirling and pipetting up and down to ensure proper pelleting. Swirl the tube briefly and place in the 37 degrees incubator O/N. Fasih will check the tubes Tuesday morning for OD verification.