Imperial/Wet Lab/Protocols/CE1.2

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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-lutamate
**1mM dithiothreitol (DTT)
**0.05% (v/v) 2-mercaptoethanol (2-ME)

====Procedure====
'''Growing the cells'''
#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.
#Harvest cells when O.D.600 = 4.5. '''At this point, cells are at mid-log phase.'''
#Wash cells three times by suspending them in 20ml of buffer A per gram of wet cells.
#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'''
#Suspend thawed cells in 12.7ml of buffer B per 10g of wet cells.
#Disrupt cells in a French press cell at a constant pressure of 20,000psi.'''This is about 140,000kPa.'''

'''Retaining the cell extract'''
#Centrifuge the crude lysate at 12,000RCF for 10min at 4°C.
#Carefully remove the top layer of the supernatant (lipid layer) and the pellet.
#Briefly pre-incubate the recovered supernatant at 37°C for 30min.
#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.