Editing In vitro translation (section)

== Preparation of the S30-lysate with expressed T7 RNA polymerase (optional) ==

In order to have T7 RNAP in your final cell-free reaction, you can use a commercial T7 RNAP preparation or use T7 RNAP purified in your lab. Note that increasing amounts of glycerol might inhibit the cell-free reaction, therefore only highly concentrated T7 RNAP should be used (~1 KU/µl) in the cell-free reaction.

However, why use a protein purified from&nbsp;''E. coli''&nbsp;in order to add it to&nbsp;''E. coli''&nbsp;lysate? It is more convenient if you use a strain which can express the T7 RNAP gene upon, e.g. strains containing the DE3 lysogen with a copy of T7 RNAP like BL21(DE3) or a strain with a T7 RNAP expression vector. You can use the same protocol as above with a small modification:

*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 0.8 to 1 at 37°C, 150-180 rpm.
*Induce the T7-RNAP production by adding 1 ml 0.5 M IPTG and grow till OD<sub>600</sub>&nbsp;3 to 4

The subsequent steps are identical with the first protocol.

Note: You have to find an optimum ratio for the standard and T7 lysate in order to have highest productivity in the reaction mix (the T7 lysate is generally less productive than the standard lysate, thus you shouldn't use only the T7 lysate). A ratio of 1:5 or 1:10 (T7:standard) should be tested initially.

'''Reaction buffers'''

The final reaction mix is set up according to following table (note: there are varying concentrations for some of the components in different publications, we have listed the ones that we found to work best in our hands, you can try other variants as well)

'''Energy Mix (3.2x):'''

(Can be stored at -20° for several months)

{| border="0"
|-
| <div>'''stock'''</div>
| <div>'''final conc.'''</div>
| <div>'''Vol. [µl]'''</div>
|-
| DTT
| <div>1000</div>
| <div>mM</div>
| <div>1,7</div>
| <div>mM</div>
| <div>19,7</div>
|-
| NTPs *
| <div>30</div>
| <div>mM</div>
| <div>1,2</div>
| <div>mM</div>
| <div>464,0</div>
|-
| cAMP
| <div>100</div>
| <div>mM</div>
| <div>0,65</div>
| <div>mM</div>
| <div>75,4</div>
|-
| Creatine phosphate (CP)
| <div>3000</div>
| <div>mM</div>
| <div>80</div>
| <div>mM</div>
| <div>309,3</div>
|-
| tRNA (E. coli)
| <div>87,5</div>
| <div>mg/ml</div>
| <div>0,175</div>
| <div>g/l</div>
| <div>23,2</div>
|-
| K-Glutamate
| <div>4000</div>
| <div>mM</div>
| <div>200</div>
| <div>mM</div>
| <div>580,0</div>
|-
| PEG 8000
| <div>30</div>
| <div>%</div>
| <div>2</div>
| <div>%</div>
| <div>773,3</div>
|-
| EM Buffer
| <div>10x</div>
| <div>1160,0</div>
|-
| ddH2O
| <div>220,0</div>
|-
| '''Total'''
| <div>'''3625,0'''</div>
|}

*NTP stock solution contains 30 mM ATP and 20 mM each CTP, GTP und UTP (final concentration of GTP/CTP/UTP of 0.8 mM).

'''10 x EM buffer:'''<br/>(for 10 ml final volume)

{| border="0"
|-
| <div>'''stock'''</div>
| <div>'''final conc.'''</div>
| '''Vol. [µl]'''
| <div>µl</div>
|-
| HEPES/KOH, pH7.5
| <div>1380</div>
| <div>mM</div>
| <div>550</div>
| <div>mM</div>
| 3985,5
| <div>µl</div>
|-
| Folinic acid
| <div>1</div>
| <div>Mg/ml</div>
| <div>350</div>
| <div>µg/ml</div>
| 3500,0
| <div>µl</div>
|-
| NH<sub>4</sub>OAc
| <div>5000</div>
| <div>mM</div>
| <div>280</div>
| <div>mM</div>
| 560,0
| <div>µl</div>
|-
| Mg(OAc)<sub>2</sub>
| <div>1000</div>
| <div>mM</div>
| <div>110</div>
| <div>mM</div>
| 1100,0
| <div>mg</div>
|-
| NaN<sub>3</sub>
| <div>0,5</div>
| <div>%</div>
| 50,0
| <div>µl</div>
|-
| ddH<sub>2</sub>O
| 854,5
| <div>µl</div>
|-
| '''Total'''
| '''10000,0'''
|}