Name:

Date:

E.coli strain to make competent:

Materials and Equipment

Please book the Equipment listed below via our online booking system PPMS or by scanning the QR code on the system itself. You don't have a PPMS account yet? Feel free to contact us. 
Please ensure the following is at hand and prepared in sufficient amounts.

  • S1 Eppendorf 5920R
  • Incubator Innova S441
  • S1 Laminar Flow
  • 30mL TFB1 for 100mL LB
  • 2mL TFB2 for 100mL LB
  • 100mL LB per strain
  • selection marker if needed:
  • 2x 50mL Falcon Tube per 100mL LB, chilled in -80°C
  • Eppis
  • liquid Nitrogen
  • glycerol stock of strain of interest:
  • original vial of strain of interest:


Buffers


TFB1

500mL

1000mL

30 mM K-acetate

1.47g

2.94g

50 mM MnCl

4.94g

9.88g

100 mM RbCl

6.04g

12.08g

10 mM CaCl2

0.735g

1.126g

15 % Glycerol (v/v)

75mL

150mL

table 01 - TFB1 buffer composition
set pH to 5.8 with 0.2 M acetic acid, sterilize with filtering



TFB2

500mL

10 mM MOPS

1.07 g

75 mM CaCl2

5.5 g

10 mM RbCl

0.6 g

15 % Glycerol (v/v)

75 mL

table 02 - TFB2 buffer composition
set pH to 7.0 with NaOH, sterilize with filtering

keep buffers at 4°C for storage

Day 1, date:

  • inoculate 10mL LB in 50mL flasks from original E.coli stock or from glycerol stock
  • add 10µL of your desired selection marker
  • please note: if there is no selection marker needed, work as sterile as possible
  • growth at 37°C, 180 RPM overnight


Day 2, date:

  • inoculate 100mL LB with ___ mL overnight culture (e.g. 3-5mL)
  • incubate at 37°C, 180 RPM until OD600 = 0.5 – 0.6
  • please note: depending on the strain this may take 1.5 – 2.5h


time






OD600









  • transfer to 50mL Falcon Tubes, pre-chilled in -80°C
  • 1st centrifugation at 3484xg, 20min. at 4°C
  • resuspend pellets carefully in 15mL ice cold TFB1 buffer each (no vortexing)
  • total volume should be 30mL from 100mL culture
  • incubate on ice for 10 min.
  • 2nd centrifugation at 23484xg, 20min. at 4°C
  • resuspend pellets carefully in 1mL ice cold TFB2 buffer each (no vortexing)
  • aliquot in 50 µL portions 1.5 mL Eppis, pre-chilled in -80°C, and freeze in liquid Nitrogen
  • store at -80°C
  • perform test transformation to see if procedure was successful (also include a blank
      transformation)



Day 3, date:

  • check plates