Procedure

day1:

Note: If glycerol cultures of transformed cells are available go directly to day2.

1. Make competent cells
2. Transform competent cells

day2:

1. Put 5 ml filter sterile LB into universal tube plus appropriate antibiotics
2. Inoculate 5mL culture with  colony picked from plate or scratched off glycerol culture
   Grow cells during the day
3. Prepare 100mL sterile filtered LB  in 0.5L shaker flask and autoclave
   Add appropriate antibiotic before inoculation
   Take 1 mL from day culture to inoculate 100ml LB night culture

Things to have ready before starting:

• plates or glycerol cultures
• sterile LB
• sterile filters and plastic syringe of appropriate size
• small plastic tubes ("universals")  to grow cells
• appropriate antibiotic
• 100 mL in 0.5 L shaker flask, all together autoclaved, antibiotic added afterwards

day3:

1. Harvest cells from night culture in autoclaved 0.5L buckets
2. Centrifuge in GS3 rotor 10-15' @ 5000 rpm
3. Resuspend cell pellet gently in 10 mL LB (use vortex, avoid too much pipetting)
4. Inoculate 0.5L of expression medium with 1-2 mL of cell suspension
5. Grow cells in shakers at appropriate temperature and speed
   (usually 37C and 200 rpm, make sure temperature is really there)
   Monitor growth by measuring OD @ 600nm (in steps of ~1h)
6. Induce expression between 0.7-1.0 OD with 1/1000 0.75 M IPTG
   (0.89 g/ 5 mL fresh stock, sterile filtered)
7. Continue growth for another 3-5h
8. Harvest cultures in 0.5L buckets
9. Centrifuge 15' @ 5000 rpm in GS3 rotor, discard supernatant
10. Take up cells in 6 mL per L medium appropriate buffer
11. Freeze cell suspension in -20 freezer

Note: it is also possible to freeze the cell pellets in the 0.5L buckets after removing the medium

Things to have ready before starting:

• thaw frozen antibiotic aliquod(s) on time.
• autoclaved 0.5L centrifuge bucket, preferably prepared evening of day 2
• sterile filters and plastic syringe of appropriate size.
• 500 mL LB in 2.0 L shaker flask, all together autoclaved, antibiotic added afterwards. Prepare on day 2, put into departmental autoclave on 2nd floor, room 215 (preferably no later than 3pm).
• book shaker as early as possible.
• book centrifuge as early as possible (left sorvall, GS3 rotor).
• switch on small photometer in big lab.
• look for plastic cuvettes.
• prepare IPTG solutiuon.
• look for 0.5 L buckets.
• have appropriate buffer ready on ice to take up cells.