Difference between revisions of "Team:Freiburg/Protocols/Cellfex"
(Created page with " {{Freiburg/CSS}} {{Freiburg/Menubar}} {{Freiburg/LabjournalCSS}} {{Freiburg/wiki_content_start}} <html> <!-- Labjournal content goes in here --> <div class="content_box"> <...") |
|||
Line 112: | Line 112: | ||
</li> | </li> | ||
<li class="level1"><div class="li"> amount of lysate used can be varied (here 45%)</div> | <li class="level1"><div class="li"> amount of lysate used can be varied (here 45%)</div> | ||
+ | </li> | ||
+ | <li class="level1"><div class="li"> the used chemicals should have a high grade of purity level of purity</div> | ||
</li> | </li> | ||
<li class="level1"><div class="li"> feed: adding 10 mM Mg(OAc) every 20 minutes can increase reaction output profoundly</div> | <li class="level1"><div class="li"> feed: adding 10 mM Mg(OAc) every 20 minutes can increase reaction output profoundly</div> |
Revision as of 13:14, 14 September 2015
![](https://static.igem.org/mediawiki/2015/8/85/Freiburg_wiki_background_2_parted_left.png)
![](https://static.igem.org/mediawiki/2015/f/f4/Freiburg_wiki_background_2_parted_right.png)
Protocol Cell-free expression
How to express proteins cell-free from DNA templates
material: Mastermix, lysate, nuclease free water and DNA template
duration: Preparation 30-45 min + running time
Mastermix
Component | stock concentration | amount in 50 µl reaction [µl] |
---|---|---|
Creatine phosphate | 80 mM | 4 |
Potassium glutamate | 200 mM | 3.7 |
HEPES-KOH pH 7.5 | 55 mM | 2.75 |
Folinic acid | 35 µg/ml | 1.75 |
Amino acids | 2mM | 1.25 |
E. coli tRNA | 175 µg/ml | 1.14 |
PEG4000 | 2% | 1 |
DTT | 1,7 mM | 0.85 |
ATP | 1,2 mM | 0.6 |
MgOAc | 11 mM | 0.55 |
CTP | 0.8 mM | 0.4 |
GTP | 0.8 mM | 0.4 |
UTP | 0.8 mM | 0.4 |
cAMP | 0.65 mM | 0.325 |
Creatine phosphokinase | 80 mM | 0.175 |
Final reaction
Component | stock concentration | amount in 50 µl reaction [µl] |
---|---|---|
Master mix | - | 17.68 |
Lysate | - | 22.5 |
DNA | at least 217 ng/µl | 3250 - 5000 ng |
H2O (nuclease free) | - | up to 50 µl |
Implementation
- incubate 2-4 hrs at 37 °C in autoclaved tubes or 96 well plate (low bind)
Remarks
- add creatine phosphokinase last to mastermix
- work quickly, sterile and as nuclease free as possible (on ice)
- add DNA last to start reactions simultaneously
- amount of lysate used can be varied (here 45%)
- the used chemicals should have a high grade of purity level of purity
- feed: adding 10 mM Mg(OAc) every 20 minutes can increase reaction output profoundly
Reaction is adapted from the European Molecular Biology Laboratory (EMBL), Heidelberg, Germany