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From 2013.igem.org

Contents 1 301 1.1 main purpose 1.2 Procedure 1.3 Results 2 115 2.1 main purpose 2.2 Who was in the lab 2.3 Procedure 2.4 Preparation of SOB medium 2.5 Preparation of CCMB80 buffer (500 ml) 2.6 Preparation of LB agar 2.7 Conclusion from today 2.8 Cultivation of TOP10 cells in SOB

301

main purpose

We want to view our cells throught a fluorescent microscope, in order to see if they have expressed GFP and RPF.

Hopefully we will see GFP in the periplasm and RFP inside the cell.

Setup an overnight culture of the 2 successful transformants so we can both MidiPrep and isolate periplasmic proteins tomorrow.

Procedure

Results

We managed to see expressed GFP and RFP

115

main purpose

Preparation for making competent cells based on the protocol ([1]).

Who was in the lab

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Henrike, Ariadni, Kashia, Natalia

Procedure

Preparation of SOB medium

Total volume 1 L

• 5 g yeast extract
• 20.05 g tryptone
• 0.58 g NaCl
• 0.19 g KCl
• 2.41 g MgSO4

Preparation of CCMB80 buffer (500 ml)

• KOAc :5 ml (0.98 gr)
• CaCl2.2H2O (5.9 g)
• MnCl2.4H2O (2 g)
• MgCl2.6H2O (1 g)
• 10% glycerol (50 ml)

Adjustment of pH (6.37) with 0.1 HCl

Preparation of LB agar

Conclusion from today

Cultivation of TOP10 cells in SOB

• 1 loop of TOP10 cells (from 14.06.2013)
• 10 ml of SOB medium

Incubate for 16 hours in 24 degrees and 37 degrees.