Imperial/Wet Lab/Protocols/CE1.2

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====Procedure====
====Procedure====
'''Growing the cells'''
'''Growing the cells'''
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#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.'''
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#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.'''
#Add 1mM IPTG to cell culture to express T7 RNA polymerase.
#Add 1mM IPTG to cell culture to express T7 RNA polymerase.
#Harvest cells when O.D.600 = 4.5. '''At this point, cells are at mid-log phase.'''
#Harvest cells when O.D.600 = 4.5. '''At this point, cells are at mid-log phase.'''

Revision as of 01:57, 27 October 2007


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.
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