Team:Groningen/Labwork/29 August 2013

From 2013.igem.org

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<h2>Friso</h2>
<h2>Friso</h2>
 +
<b>Testing Assembly of spider silk</b>
 +
<br><i>(over the past three weeks, will put it at each day separately and more detailed soon)</i>
 +
<br>
 +
<br>Made large culture (200 ml) E. coli from Utah 2012
 +
<br>
 +
<br>After 24h, removed medium, stored cells (about 1.2 grams wet cell weight)
 +
<br>
 +
<br>Added lysis buffer and lysed the cells by sonication
 +
<br>
 +
<br>Removed cell rests and insoluble proteins by sedimentation 12000 rpm
 +
<br>
 +
<br>Boiled the protein mixture
 +
<br>
 +
<br>Removed precipitated proteins by sedimentation 12000 rpm
 +
<br>
 +
<br>Impure solution of silk proteins in water: formed film by evaporation at 55 *C
 +
<br>
 +
<br>Treated film with 1M Potassium phosphate, evaporated at 55 *C
-
'''Testing Assembly of spider silk'''
+
<h2>Sebas</h2>
-
''(over the past three weeks, will put it at each day separately and more detailed soon)''
+
- miniprepped pX vector
 +
<Br>- Digested pX with SpeI*SdaI (fermentas) in SdaI buffer with 2:1 SpeI:SdaI.
 +
<Br>- Gel purified the digested backbone.
 +
<Br>
 +
<Br>- digested BBa_K10805000 (S1) and BBa_K1085008 (S13)with EcoRI*SpeI and XbaI*PstI respectively 1h 37degrees.
 +
<Br>- heat inactivated the sample and ligated S1+S13 for 1h at 25degrees in water bath
 +
<Br>- heat inactivated the T4 ligase and digested product with XbaI.
 +
<Br>- Heat inactivated XbaI and ligated the insert (X-S1+S13-P) with the backbone (SpeI-pX-SdaI) in a 3:1 molar <Br>ratio. 
-
Made large culture (200 ml) E. coli from Utah 2012
 
-
After 24h, removed medium, stored cells (about 1.2 grams wet cell weight)
 
-
 
-
Added lysis buffer and lysed the cells by sonication
 
-
 
-
Removed cell rests and insoluble proteins by sedimentation 12000 rpm
 
-
 
-
Boiled the protein mixture
 
-
 
-
Removed precipitated proteins by sedimentation 12000 rpm
 
-
 
-
Impure solution of silk proteins in water: formed film by evaporation at 55 *C
 
-
 
-
Treated film with 1 M Potassium phosphate, evaporated at 55 *C
 
</div>
</div>

Latest revision as of 10:04, 3 September 2013

Inne


09:15 plates were taken out of incubators.
10:12 the plates were put back into incubators.
16:25 plates taken out of incubators.

Claudio

The ligation reaction containing S15+S4+pSB1C3 and the positive control (only pSB1C3) were transformed in E. Coli DH5α.
The competent cells were plated on LB + Cm and incubated at 37°C overnight.

Sander

made a plate with liquid SGG medium and a plastic object partially submerged and placed in a 30 C stove.

Friso

Testing Assembly of spider silk
(over the past three weeks, will put it at each day separately and more detailed soon)

Made large culture (200 ml) E. coli from Utah 2012

After 24h, removed medium, stored cells (about 1.2 grams wet cell weight)

Added lysis buffer and lysed the cells by sonication

Removed cell rests and insoluble proteins by sedimentation 12000 rpm

Boiled the protein mixture

Removed precipitated proteins by sedimentation 12000 rpm

Impure solution of silk proteins in water: formed film by evaporation at 55 *C

Treated film with 1M Potassium phosphate, evaporated at 55 *C

Sebas

- miniprepped pX vector
- Digested pX with SpeI*SdaI (fermentas) in SdaI buffer with 2:1 SpeI:SdaI.
- Gel purified the digested backbone.

- digested BBa_K10805000 (S1) and BBa_K1085008 (S13)with EcoRI*SpeI and XbaI*PstI respectively 1h 37degrees.
- heat inactivated the sample and ligated S1+S13 for 1h at 25degrees in water bath
- heat inactivated the T4 ligase and digested product with XbaI.
- Heat inactivated XbaI and ligated the insert (X-S1+S13-P) with the backbone (SpeI-pX-SdaI) in a 3:1 molar
ratio.