Team:Marburg:Project:Notebook

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Notebook: March

17.03.2014

1.1 Overnight Preculture

2x5 mL LB medium were inoculated with 50 µL DH5Alpha, aliquoted for a preculture and incubated overnight (16,5h) at 37°C.

18.03.2014

1.2 Main Culture

250ml were inoculated with 5ml 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 3ml HTP buffer. In the end, 225µl DMSO was added to an end concenctration of 6-7%. The 50µl aliquots were frozen in N2(l) and stored at -80°C.

26.03.2014

5.1 Testing Competence and Transformation of DH5Alpha

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

7.1 E. coli DH5Alpha with PSG1164: Overnight-cultures

50ml LB-medium (with ampicilin) were inoculated with E.coli BL21 PlyS and grown overnight.

27.03.2014

5.2 Results

Plates from 26.03.2014

LB-plate growth
canamycin-plate no growth
ampicilin-plate no growth
ampicilin-plate 2 trafo successfull growth
7.2 Inoculation

Inoculation of LB-plates with E.Coli DH5Alpha with PSG1164.

7.3 Miniprep

Miniprep according to the miniprep kit protocol (Omega) with 2x6ml 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 - NcolI (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 Cutsmarkt
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. 5µl marker and 10µl of the samples are loaded onto the SDS-gel.

Results: