User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/15

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Finishing Expression of Wildtype Hemoglobin

  1. Spin the Bl21(DE3) + wt Asc Hb induced cells (from overnight growth) at 4500rpm at 4°C.
  2. Resuspend cells in 25mM Tris, 50mM NaCl, pH 8.
  3. Freeze the cells in liquid nitrogen.
  4. Store at -20°C.

Cutting with HindIII for Cloning

This was done with the pQE-80-L-Kan.

  1. Mix 2μL of pQE-80-L-Kan with 5μL of 10x NEBuffer2, 25μL of sterile dH2O, and 5μL of HindIII enzyme.
  2. Incubate for two hours at 37°C.
  3. Incubate for 20 minutes at 65°C.

Making Media for Expression

  • Make 4 1L LB broths: each is 25g LB (w/o NaCl) + 10g NaCl with 1L of dH2O
  • Make 4 50mL LB broths: each is 1.25g with 50mL of dH2O
    • Autoclave all of these broths
    • Also, autoclaved a dry cycle today.

Cutting pQE80-L-Kan with BamHI for Cloning (Double Digest)

  • This will be done with the already HindIII digested pQE80-L-Kan from this morning.
  • What NEB suggests for double digests can be found here: [1]

The digest:

  1. Mix 45μL of this morning's HindIII digest product with 5μL of BamHI-HF (HF=high fidelity).
  2. Incubate for two hours at 37°C.
  3. Store at -20°C.