Finishing Expression of Wildtype Hemoglobin
- Spin the Bl21(DE3) + wt Asc Hb induced cells (from overnight growth) at 4500rpm at 4°C.
- Resuspend cells in 25mM Tris, 50mM NaCl, pH 8.
- Freeze the cells in liquid nitrogen.
- Store at -20°C.
Cutting with HindIII for Cloning
This was done with the pQE-80-L-Kan.
- Mix 2μL of pQE-80-L-Kan with 5μL of 10x NEBuffer2, 25μL of sterile dH2O, and 5μL of HindIII enzyme.
- Incubate for two hours at 37°C.
- 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:
- Mix 45μL of this morning's HindIII digest product with 5μL of BamHI-HF (HF=high fidelity).
- Incubate for two hours at 37°C.
- Store at -20°C.
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