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cell-free-prep.md

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Day 0

S30 buffer (A)

  • 10 mM Tris-acetate buffer
  • 14 mM Mg(OAc)2
  • 60 mM KOAc
  • 1 mM DTT
  • 0.5 ml/l 2-mercaptoethanol
  • pH 8.2

S30 buffer (B)

  • 10 mM Tris-acetate buffer
  • 14 mM Mg(OAc)2
  • 60 mM KOAc
  • 1 mM DTT
  • pH 8.2

Preincubation buffer

  • 293 mM Tris-OAc buffer
  • pH 8.2
  • 9.2 mM Mg(OAc)2
  • 13.2 mM ATP (pH 7.0)
  • 84 mM PEP (pH 7.0)
  • 4.4 mM DTT
  • 40 μM of each of the 20 amino acids!
  • 6.7 U/ml PK (Sigma P7768)

Batch reaction mix

30 uL aliquot with 6.7 μg/ml of the pK7-CAT plasmid

  • 55 mM Hepes-KOH buffer (pH 7.5) containing
  • 1.7 mM DTT
  • 1.2 mM ATP (pH 7.0)
  • 0.8 mM each of CTP (pH 7.0)
  • GTP (pH 7.0)
  • UTP (pH 7.0)
  • 80 mM CP
  • 250 μg/ml CK
  • 4% PEG 8000
  • 0.64 mM 3',5'-cyclic AMP
  • 68 μM L(–)-5-formyl-5,6,7,8-tetrahydrofolic acid
  • 175 μg/ml E. coli total tRNA
  • 210 mM potassium glutamate
  • 27.5 mM ammonium acetate
  • 10.7 mM magnesium acetate
  • 1.0 mM of each of the 20 amino acids
  • 93 μg/ml T7 RNA polymerase
  • 9.0 μl S30 extract.

Dialysis mix

internal solution

  • all of the components used for the batch mix
  • 0.05% sodium azide

external solution

  • the components of the internal solution except for CK, the plasmid vector, the T7 RNA polymerase, E. coli total tRNA, and the S30 extract

Day 1

Growth and washing

  • BL21 CP strain was grown at 37 °C in six 2-l baffled flasks, each with 500 ml 2 􏰁 YT medium con- taining 34 μg/ml Cm, by circular shaking at 160 rpm.

  • Cells were harvested in the mid-log phase (OD600 􏰅 3, usually 3–4 h)

  • washed three times with the S30 buffer (A)

  • At this stage, we used a Polytron cell homogenizer (Kinematica AG, PT-MR3100) to facilitate resuspension of the cell pellets.

Storage

  • flash freeze in LN2 for at least 2 min
  • stored at –80 °C for 1–3 days

Yield

In a typical case, about 16 g of loosely packed cells are obtained from a 3 l culture.

Day 2

Resuspension and disruption

  • The frozen cells were thawed in 50 ml of the S30 buffer (A)

  • and then collected by centrifugation at 16,000􏰁g for 30 min at 4°C

  • Cells (7g) were suspended in 8.9 ml of the S30 buffer (B) and disrupted with 22.7 g of glass beads (B. Braun Melsungen AG 854–150/7, 􏰆 􏰃 0.17–0.18 mm) by the Multi-beads Shocker Type MB301 (Yasui Kikai, http://www.yasuikikai.co.jp) with the program: 30 sec on, 30 sec off, 30 sec on, 30 sec off, 30 sec on

  • The preparation was centrifuged twice at 30,000 􏰁 g for 30 min at 4 °C, to remove the glass beads.

Dialysis (4 hours)

  • The resultant supernatant (4.9 mL) was incubated at 37 °C for 80 min with 0.3 volume of the preincubation buffer (see recipe).

  • The incubated sample was dialyzed 4 times against 50 volumes of the S30buffer (B) for 45 min each at 4 °C

  • centrifuged at 4,000 􏰁 g for 10 min at 4 °C to obtain the supernatant (the S30 extract).

Storage

  • dispensed into 0.2–2.0 ml aliquots, immediately and completely frozen by being immersed in liquid nitrogen for at least 2 min

  • store in liquid nitrogen or below –80 °C. The S30 extract remains active for at least 1 year if stored in liquid nitrogen.

Day 3: Expression

  • The batch reaction mixture was incubated at 37 ̊C for 1 h.

  • The internal solution was dialyzed in a dialysis tube (Spectra/Por 7 MWCo:15,000, Spectrum) against the external solution at 30 °C for 8 h with shaking.