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Wet Lab: Protocols: Preparation of E. coli S12 Extract

Day 1

Equipment

• 37°C shaking incubator
• 1L conical flasks x 3
• Pipette fillers + pipettes (5ml, 10ml and 25ml)
• Spectrometer + cuvettes
• Weighing scale
• Centrifuge + 150ml centrifuge tubes
• Pipettes + pipette tips (20µl, 200µl and 1000µl)

Reagent

• 2xYT medium
• IPTG
• Buffer A
  • 10mM Tris-acetate (pH 8.2)
  • 14mM Mg-acetate
  • 60mM K-glutamate
  • 1mM dithiothreitol (DTT)
  • 0.05% (v/v) 2-mercaptoethanol (2-ME)

Procedure

Growing the cells

1. Grow E. coli strain BL21 (DE3) cells at 37°C in 3L of 2xYT medium till O.D.600 = 0.6.Ensure vigorous agitation and aeration.
2. Add 1mM IPTG to cell culture to express T7 RNA polymerase.
3. Harvest cells when O.D.600 = 4.5. At this point, cells are at mid-log phase.
4. Wash cells three times by suspending them in 20ml of buffer A per gram of wet cells.
5. Centrifuge and weigh the wet cell pellets before storing them at -80°C.

(Note: The cells may take more than 1 day to grow to O.D.600 = 4.5.)

Day 2

Equipment

• Pipette filler + pipettes (5ml, 10ml and 25ml)
• Weighing scale
• French press + French press cell
• Centrifuge + 50ml centrifuge tubes
• Pipette + pipette tips (20µl, 200µl, 1000µl)
• 37°C shaking incubator
• Dialysis membrane with molecular weight cut-off of 10,000
• Magnetic stirrer
• 4°C cold room

Reagent

• Buffer B
  • 10mM Tris-acetate (pH 8.2)
  • 14mM Mg-acetate
  • 60mM K-glutamate
  • 1mM DTT

Procedure

Lysing the cells

1. Suspend thawed cells in 12.7ml of buffer B per 10g of wet cells.
2. Disrupt cells in a French press cell at a constant pressure of 20,000psi.This is about 140,000kPa.

Retaining the cell extract

1. Centrifuge the crude lysate at 12,000RCF for 10min at 4°C.
2. Carefully remove the top layer of the supernatant (lipid layer) and the pellet.
3. Briefly pre-incubate the recovered supernatant at 37°C for 30min.
4. Divide resulting S12 extract into small aliquots and store at -80°C.

Notes

• Total time required: ~ 3 days.
• The protocol is based on [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T3C-4K242VK-1&_user=10&_coverDate=12%2F01%2F2006&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=fbf36c742708c667b5c4481856ca22b5 Simple procedures for the construction of a robust and cost-effective cell-free protein synthesis system] by Kim DM et al.
• We did not prepare any E. coli S12 extract because of the lack of reagents and limited budget.