Revision as of 20:42, 15 October 2014

Notebook: March

17.03.2014

2x5 mL LB medium were inoculated with 50 µL DH5α, aliquoted for a preculture and incubated overnight (16,5h) at 37 °C.

250 mL were inoculated with 5 mL of preculture and incubated until they reached an OD of 0,5. After centrifugation, the cell pellet was washed with HTP buffer and resuspended in 3 mL HTP buffer. In the end, 225 µL DMSO was added to an end concentration of 6-7%. The 50 µL aliquots were frozen in N₂(l) and stored at -80 °C.

26.03.2014

5.1 Testing Competence and Transformation of DH5α

1 µL plasmid with a Amp-resistance was put on ice for 10 min. A heat shock was induced for 45 s at 42 °C. The sample was put on ice for another 10 min and then incubated in 200 µL LB for 1 h before it was plated onto LB-, ampicillin- and canamycin-plates.

7.1 E. coli DH5α with PSG1164: Overnight-cultures

5 mL LB-medium (with ampicillin) were inoculated with E.coli BL21 pLysS and grown overnight.

27.03.2014

5.2 Results

Plates from 26.03.2014

LB-plate	growth
canamycin-plate	no growth
ampicillin-plate	no growth
ampicillin-plate 2	trafo successful growth

7.2 Inoculation

Inoculation of LB-plates with E.Coli DH5α with PSG1164.

7.3 Miniprep

Miniprep according to the miniprep kit protocol (Omega) with 2x6 mL preculture.

DNA-concentration:

1. 233,2 ng/µL
2. 261,3 ng/µL

7.5 Test Digestion

Tested enzyme	Compatible with Ncol	Compatible with Ncol-HF
AvrII	100	100
KpnI / HF	50	50*
ApaI / HF	100	100
XhoI / HF	100	100
SalI / HF	10	10/100
ClaI / HF	100	100
HindIII / HF	50*/100	50/100
EcoRV / HF	10*/100	10/100
EcoRI / HF	50*/100	50*100
PstI / HF	50*/100	50*100

7.6 Digestion of PSG1164

Digestion scheme:

[µL]	PSG1164 - uncut	PSG1164 - NcoI (20kU/mL)	PSG1164 - EcoRI-HF
DNA	261,3 ng/µL ca. 1 µg → 3,5 µL	261,3 ng/µL ca. 1 µg → 3,5 µL	261,3 ng/µL ca. 1 µg → 3,5 µL
Enzyme 1	-	Ncol 0,5	-
Enzyme 2	-	-	EcoRI-HF 0,5
Buffer	-	10x Tango Buffer 1,5	10x Cutsmart 1,5
H₂O	11,5	9,5	9,5
Total Volume	15	15	15
Loading Dye (6x)	2,5	2,5	2,5

Incubate samples for 60 min at 37 °C, then induce a heat shock for 2 min at 45 °C. 5 µL marker and 10 µL of the samples are loaded onto a 1% agarose gel.

Results:

The plasmid digested with NcoI showed two bands, what means, it had two restriction sites. pSG1164 digested with EcoRI looked like it was linearized.

@@ Line 52: / Line 52: @@
      <legend><a name="exp7.1">7.1 <i>E. coli</i> DH5&alpha; with PSG1164: Overnight-cultures</a></legend>
 	<div class="exp-content">
-		<p>50 mL LB-medium (with ampicillin) were inoculated with <i>E.coli</i> BL21 pLysS and grown overnight.</p>
+		<p>5 mL LB-medium (with ampicillin) were inoculated with <i>E.coli</i> BL21 pLysS and grown overnight.</p>
      </div>
 </fieldset>
@@ Line 85: / Line 85: @@
 	      </tr>
 	  </table>
+	  <img src="
      </div>
 </fieldset>
@@ Line 229: / Line 230: @@
          </tr>
      </table>
-	<p>Incubate samples for 60 min at 37 &deg;C, then induce a heat shock for 2 min at 45 &deg;C. 5 &micro;L marker and 10 &micro;L of the samples are loaded onto a 1% agarose gel</p>
+	<p>Incubate samples for 60 min at 37 &deg;C, then induce a heat shock for 2 min at 45 &deg;C. 5 &micro;L marker and 10 &micro;L of the samples are loaded onto a 1% agarose gel.</p>
      </div>
 	<div class="exp-results">
      <h3>Results:</h3>
      <!-- exp7.7 -->
-		<img src="https://static.igem.org/mediawiki/2014/5/51/2014-03-07_PSG1164.jpg" width="30%" />
+		<img src="https://static.igem.org/mediawiki/2014/3/34/MR_20140327_restriction_pSG1164_NcoI_and_EcoRI.jpg" width="30%" />
+		<p>The plasmid digested with <i>Nco</i>I showed two bands, what means, it had two restriction sites. pSG1164 digested with <i>Eco</i>RI looked like it was linearized.</p>
      </div>
 </fieldset>

Team:Marburg:Project:Notebook

From 2014.igem.org