Team:Marburg:Project:Notebook

From 2014.igem.org

Notebook: March

17.03.2014

1.1 Overnight Preculture

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

18.03.2014

1.2 Main Culture

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.