Difference between revisions of "Team:Freiburg/Protocols/prepLysate"
| Line 57: | Line 57: | ||
<div class="table sectionedit3"><table class="inline"> | <div class="table sectionedit3"><table class="inline"> | ||
<tr class="row0"> | <tr class="row0"> | ||
| − | <th class="col0">Component </th><th class="col1"> stock concentration </th><th class="col2"> | + | <th class="col0">Component </th><th class="col1"> stock concentration </th><th class="col2">endconcentration [µl]</th> |
</tr> | </tr> | ||
<tr class="row1"> | <tr class="row1"> | ||
| − | <td class="col0"> | + | <td class="col0"> Tris Ac, pH 7.6</td><td class="col1"> - </td><td class="col2"> 300mM </td> |
</tr> | </tr> | ||
<tr class="row2"> | <tr class="row2"> | ||
| − | <td class="col0"> | + | <td class="col0"> Mg(OAc)2 </td><td class="col1"> - </td><td class="col2"> 10 mM </td> |
</tr> | </tr> | ||
<tr class="row3"> | <tr class="row3"> | ||
| − | <td class="col0"> | + | <td class="col0"> ATP </td><td class="col1"> at least 217 ng/µl </td><td class="col2"> 10mM </td> |
</tr> | </tr> | ||
<tr class="row4"> | <tr class="row4"> | ||
| − | <td class="col0"> | + | <td class="col0"> phosphoenol pyruvate (PEP) </td><td class="col1"> - </td><td class="col2"> 80mM </td> |
| + | </tr> | ||
| + | <tr class="row2"> | ||
| + | <td class="col0"> Amino Acid mix </td><td class="col1"> - </td><td class="col2"> 5mM </td> | ||
| + | </tr> | ||
| + | <tr class="row3"> | ||
| + | <td class="col0"> pyruvate kinase</td><td class="col1"> at least 217 ng/µl </td><td class="col2"> 3250 - 5000 ng </td> | ||
| + | </tr> | ||
| + | <tr class="row4"> | ||
| + | <td class="col0"> DEPC-H2O </td><td class="col1"> - </td><td class="col2"> up to 50 µl </td> | ||
</tr> | </tr> | ||
</table></div> | </table></div> | ||
Revision as of 15:56, 14 September 2015
Protocol Cell-free expression
Preparation of a Lysate for Cell-free exxpression
material:
duration: Preparation 1 week
Buffer A:
| Component | final concentration |
|---|---|
| TRIS-acetate (pH 8 | 10 mM |
| Potassium acetate | 60 mM |
| Magnesium acetate | 14 mM |
| DTT | 1 mM |
| ß-mercaptoethanol | 7 mM |
- Buffer B is the same as A, but without the ß-mercaptoethanol
- Buffer C is the same as B, but without DTT
Preincubation Buffer
| Component | stock concentration | endconcentration [µl] |
|---|---|---|
| Tris Ac, pH 7.6 | - | 300mM |
| Mg(OAc)2 | - | 10 mM |
| ATP | at least 217 ng/µl | 10mM |
| phosphoenol pyruvate (PEP) | - | 80mM |
| Amino Acid mix | - | 5mM |
| pyruvate kinase | at least 217 ng/µl | 3250 - 5000 ng |
| DEPC-H2O | - | up to 50 µl |
Implementation
- incubate 2-4 hrs at 37 °C in autoclaved tubes or 96 well plate (low bind)
Growth of Bacteria
Harvesting, washing and lysis
- cells were grown in a 2 litre chicanery flask containing 1 litre of Terrific Broth medium until reaching OD600 = 0,8
- The expression of T7 RNA polymerase then was induced by adding 0.5 ml of 1 M IPTG to the medium
- further growth until OD600 = 6.5, then cells were put on ice
- cells were pelleted at 6000 g at 4°C for 30 minutes in a fixed-angle rotor
- supernatant was decanted and the cells were washed two times in buffer A
- Again, the cells were pelleted at 5000 g for 20 min at 4°C and after-wards resuspended in 1.3 ml of buffer B
- feed: Using a french press with a 1“ diameter piston, the cells were lysed at 1000 psi (≈ 6.9 MPa)
- About 7.5 ml of lysate was obtained after pressing.
Harvesting, washing and lysis
- obtained lysate is pelleted via ultracentrifugation at 30,000 g for 30 min at 4°C
- The expression of T7 RNA polymerase then was induced by adding 0.5 ml of 1 M IPTG to the medium
- The resulting extract is mixed with the preincubation buffer in a ratio of 10:1 (lysate:buffer)
- preincubated lysate is then transferred to a 3.5 kDa dialysis tubing and desalted by dialysing it against buffer C at 4°C for 90 minutes
- after renewing the buffer, once more over night at slow stirring
- the lysate is ready for use and, after shock-freezing in liquid N2, can be stored in small ali-quots at - 80°C.
Remarks
- The Preincubation step takes care of what is called the „run off“ step, a step imple-mented in order to eliminate endogenous cellular mRNA. The removal of endog-enous mRNA bound to ribosomes/polysomes is accomplished by finishing their synthesis via the added PEP, pyruvate kinase and ATP at 37°C. The residual endogenous mRNA will then subsequently become degraded due to the high RNase content of the extract. This step is precautionally performed in the dark, because some components are slightly light sensitive.
- The lysate can be frozen again after use, but looses effectivity
Reaction is adapted from the European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
