Editing In vitro translation (section)

== &nbsp;Protocol for S30-ribosome-lysate ==

(adapted from Kigawa et al. (2004), Preparation of Escherichia coli cell extract for highly productive cell-free protein expression, J. Struct. Fun. Gen., 5, 63-68)

E. coli strain: BL21(DE3), BL21(DE3) CodonPlus RIL or variants (not pLysS)

&nbsp;around 7 ml of S30-ribosome-lysate can be isolated from 1 liter of culture&nbsp;

'''Per liter medium:'''

{| border="0"
|-
| 5.6 g
| KH<sub>2</sub>PO<sub>4</sub>
|-
| 28.9 g
| K<sub>2</sub>HPO<sub>4</sub>
|-
| 10 g
| yeast extract
|-
| 15 mg
| Thiamine
| add after sterilization
| (filtered sterile)
|-
| 40 ml
| 25% Glucose
| add after sterilization
| (filtered sterile)
|}

All buffers and stock solutions should be prepared with diethylpyrocarbonate (DEPC)-treated H2O (DNAse/RNAse-free).<br/>All stock-solution can be stored @ -20°C for weeks except creatine kinase. Creatine kinase has to be dissolved in 30 mM Glycine, pH = 9.0 + 20 mM DTT. After freezing in liquid nitrogen, it is stable @ -80°C for weeks.<br/>All individual amino acids have to be dissolved as described on package. The mixture contains 5 mM of each amino acid with a pH of 7.5 adjusted with KOH.

{| border="0"
|-
| width="15%" | <div>'''Compound buffer'''</div>
| width="14%" | <div>'''Tris/OAc, pH 8.2'''</div>
| width="7%" | <div>'''KOAc'''</div>
| width="10%" | <div>'''Mg(OAc)2'''</div>
| width="16%" | <div>'''DTT (prepare fresh)'''</div>
| width="20%" | <div>'''ß-ME (prepare fresh)'''</div>
| width="18%" | <div>'''Vol. needed for 1 litre of culture'''</div>
|-
| <div>'''Buffer 1'''</div>
| <div>10 mM</div>
| <div>60 mM</div>
| <div>14 mM</div>
| <div>1 mM</div>
| <div>7 mM</div>
| <div>150 ml</div>
|-
| <div>'''Buffer 2'''</div>
| <div>10 mM</div>
| <div>60 mM</div>
| <div>14 mM</div>
| <div>1 mM</div>
| <div>-</div>
| <div>10 ml</div>
|-
| <div>'''Buffer 3'''</div>
| <div>10 mM</div>
| <div>60 mM</div>
| <div>14 mM</div>
| <div>-</div>
| <div>-</div>
| <div>1500 ml</div>
|}

'''Preincubation Buffer: 0.75 ml needed for 1 litre of culture'''

{| border="0"
|-
| <div>'''vol. of stock'''</div>
| <div>'''stock'''</div>
|-
| 300 mM Tris Ac, pH 7.6
| 225 µl
| 1M
|-
| 10 mM Mg(OAc)<sub>2</sub>
| 7.5 µl
| 1M
|-
| 10 mM ATP
| 75 µl
| 100mM
|-
| 80 mM phosphoenol pyruvate (PEP)
| 60 µl
| 1M
|-
| 5 mM DTT
| 3.75 µl
| 1M
|-
| 40 µM Amino Acid mix
| 6 µl
| 5 mM
|-
| 8 U/ml pyruvate kinase
| 3 µl
| 2 U/µl
|-
| DEPC-H<sub>2</sub>O
| ad 750 µl
|}

*Prepare an overnight preculture in 10 ml medium in a 100 ml Erlenmeyer-flask @ 37°C, 250 rpm, that will have to be inoculated in 1000 ml media in a 5000 ml chicanery-flask and grown till OD600 1.5-2 at 37°C, 150-180 rpm. Care about contaminations, because no selective-marker is added!
*Centrifuge using SLA rotors at 6.000g, 30 min, 4°C, store pellet @ -80°C
*All following steps should be done on ice!
*Resuspend pellet in buffer 1 (16.6 ml buffer / gram pellet) by shaking or knocking by hand. Don´t pipet up and down!
*Centrifuge @ 5.000g, 20min, 4°C and discard supernatant.
*Resuspend pellet in buffer 1 (6.6 ml buffer / gram pellet) by shaking or knocking by hand. Don´t pipet up and down!
*Centrifuge @ 5.000g, 20min, 4°C and discard supernatant.
*Resuspend pellet in buffer 2 (1.3 ml buffer / gram pellet) by shaking or knocking by hand. Don´t pipet up and down!
*Destruct cells using French press @ 6000 psi cell pressure (390 - 400 psi using the 1´´-piston @ high ratio) for only one time!
*Pellet the lysate using ultracentrifugation @ 30.000g (around 20.000 rpm in Ti60 rotor) 30 min, 4°C (a large grey-green pellet will be formed). Transfer supernatant in new tubes and centrifuge again (a very small and clear pellet will be formed).
*Transfer supernatant by using a pipette into 15 ml tubes and incubate in preincubation buffer (1 ml buffer / 10 ml lysate) @ 37°C, 90 min by using a roller (mid-speed) in the dark (wrapped in alu foil). The lysate will be cloudy.
*Transfer lysate in high pure dialysis membrane (without heavy metals or glycerine, e.g. Roth V-Serie) mwco: 5.000 Da and dialyse @ 4°C, 1h against buffer 3 (ratio 1 ml lysate to 100 ml buffer 3), change buffer and dialyse again @ 4°C, overnight.
*Transfer lysate into 15 ml tubes and centrifuge @ 4.000g, 4°C, 10min.
*Decant the supernatant and store in aliqoutes of 500 µl.
*Shock freeze in liquid N2 and store @ -80°C.