Editing In vitro translation (section)

== Setting up a cell-free reaction ==

Depending on the protocol you are using there are variations in the reaction composition and concentration of the components. This indicates that the system is quite robust and further optimization of the cell-free reaction mix is possible.

'''Final Reaction Mix:'''

{| border="0"
|-
| width="77%" | <div>'''Component'''</div>
| width="23%" | <div>'''V [µl]'''</div>
|-
| Lysate
| <div>400,0</div>
|-
| Lysate T7
| <div>50,0</div>
|-
| Energy Mix (EM, 3.2x)
| <div>312,5</div>
|-
| Amino Acid Mix ** (2mM final each)
| <div>80,0</div>
|-
| Creatine kinase (80µg/ml final) ***
| <div>4,0</div>
|-
| Vector DNA (10µg/ml final)$
| <div>x</div>
|-
| Additives (e.g. detergents, cofactors, ions, etc)
| <div>0,0</div>
|-
| ddH2O
| <div>153,5-x</div>
|-
| '''Total'''
| <div>'''1000,0'''</div>
|}

*
*Note that the Amino Acid Mix has to be 25mM for each of the 20 AAs, some of them will not be soluble at pH 7.5-8 which should be the pH value of this mix. It is usually no problem to add it as a suspension after thorough mixing.<br/>*** Creatine kinase should be dissolved at 20mg/ml in 30 mM Glycine, pH9.0 + 20 mM DTT and stored in small aliquots at -80°C. Avoid multiple freezing and thawing.<br/>$ Vector DNA should be of high quality (Maxi-prep)