User:AndreasConstantinou/22 June 2010

From 2010.igem.org

Latest revision as of 16:05, 29 June 2010

Preparation of competent Top10 Escherichia coli cells

Continued from 21/6.
Based on the [http://openwetware.org/wiki/TOP10_chemically_competent_cells protocol of the Knight lab]

Materials

CCMB80 buffer

• 10 mM KOAc pH 7.0
• 80 mM CaCl2.2H2O
• 20 mM MnCl2
• 10 mM MgCl2.6H2O
• 10% glycerol

Procedures

1. 250 ml LB inoculated with 2 ml from ON culture
   • Grown at 28°C until an OD(600) of ≈0.3
2. Cells spun down at 3000x g for 10 min in 4°C
3. Cells resuspended in 80 ml ice-cold CCMB80 buffer and kept on ice for 20 min:
4. Cells spun down with same settings
5. Cells resuspended in 10 ml new CCMB80 and kept on ice for 20 min
6. Competent cells aliquoted in 100ul aliquots and stored in -80°C.

Testing competent cells and antibiotic agar plates

Competent cells were tested by transforming them with the Freiburg standard expression vector pEX (BBa_K243033), carrying resistance to Amp. Agar plates were tested by plating them with transformed and non-transformed cells.

Procedures

1. 1ul pEX plasmid was added to 100ul thawed, competent cells.
   • A mock-transformation was performed with dH2O.
   • Incubated on for 30 min.
2. Cells heat-shocked for 55 sec in 42°C. Cooled down on ice.
3. 900ul LB was added and cells incubated for 1 h in 37°C, 250 rpm rotary shaking.
4. Cells spun at full speed for 15 sec.
5. 900 ul was removed from the supernatant.
6. Remaining 100 ul was plated onto 100 ug/ml Amp and 25 ug/ml Cm plates and grown ON in 37°C.