Paul.xu: Created page with "== Medium and Buffers == 1. 4% lactose, 10% glucose, 80% glycerol, 2. 1 M MgSO4 3, 1000x trace metals 50 mM FeCl3, 20 mM CaCl2, 10 mM each of MnCl2 and ZnSO4, and 2..."

2020-12-29T14:49:32Z

Created page with "== Medium and Buffers == 1. 4% lactose, 10% glucose, 80% glycerol, 2. 1 M MgSO4 3, 1000x trace metals 50 mM FeCl3, 20 mM CaCl2, 10 mM each of MnCl2 and ZnSO4, and 2..."

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== Medium and Buffers ==

1.  4% lactose, 10% glucose, 80% glycerol,

2. 1 M MgSO4

3, 1000x trace metals

50 mM FeCl3, 20 mM CaCl2, 10 mM each of MnCl2 and ZnSO4, and 2 mM each of CoCl2, CuCl2, NiCl2, Na2MoO4, Na2SeO3, and H3BO3 in 60 mM HCl

4. 50x Buffer M

1.25 M Na2HPO4, 1.25 M KH2 PO4, 2.5 M NH4Cl, and 250 mM Na2SO4

== Procedure ==

Culture  E. coli BL21 Star™(DE3) (~16 h)  and ClearColi™ BL21(DE3) (~18 h)   overnight.

Dilute 1/100 in duplicate 1.5 L auto-induction media ZYM-5052 prepared to 0.05% final glucose concentration  in 2.5 L Tunair shake flasks (IBI Scientific, Peosta, IA).

Incubator  at 37 °C and 280 rpm

for BL21 Star™(DE3): Culture 5~6 hours after inoculation, OD 2.9 ~5.0 (early to mid log phase)

for ClearColi™ BL21(DE3):  Culture 5.5~8 hours after inoculation, OD =1 ~ 3.3 (early to mid log phase)

Spin cells  at 6000 g for 10 min

Discard supernatant

Wash cells with Buffer A: 10 mM Tris, 14 mM Magnesium Acetate, 60 mM Potassium Glutamate, 1 mM DTT

Free cells pellet at −80 °C.

Resuspend frozen cell paste in Buffer A (1 mL per gram cell).

Lysis with an Avestin EmulsiFlex-B15 homogenizer (Avestin, Ottawa, Canada), 3 passes at 20,000 psi.

Spin cell lysate  at 12,000 g for 10 min

Incubate supernatant at 37 °C for 30 min

Freeze and store supernatant at −80 °C.

== CFPS expression ==

50 or 70 μL in 2 mL microcentrifuge tubes 

to 12.5 ul  cell extract

12 nM plasmid DNA

10–20 mM magnesium glutamate (concentration optimized for protein yield)

1 mM 1,4-diaminobutane

1.5 mM spermidine

40 mM phosphoenolpyruvate (PEP)

10 mM ammonium glutamate

175 mM potassium glutamate

2.7 mM potassium oxalate

0.33 mM nicotinamide adenine dinucleotide (NAD)

0.27 mM coenzyme A (CoA),

1.2 mM ATP,

0.86 mM CTP,

0.86 mM GTP,

0.86 mM UTP,

0.17 mM folinic acid,

2 mM of all the canonical amino acids except glutamic acid

incubated 3 h at 37 °C and 120 rpm.

Ref:

https://www.sciencedirect.com/science/article/pii/S2405805X19300638?via%3Dihub
[[Category:Protocols]]

ClearColi Cell extract for In vitro Translation - Revision history

Paul.xu: Created page with "== Medium and Buffers == 1. 4% lactose, 10% glucose, 80% glycerol, 2. 1 M MgSO4 3, 1000x trace metals 50 mM FeCl3, 20 mM CaCl2, 10 mM each of MnCl2 and ZnSO4, and 2..."