• Add to 100ml of LB media in a 250ml flask, 1ml of overnight E. coli XL1 Blue (from a colony picked from a TET containing plate) grown in the presence of 12.5µg/ml tetracyclin in LB.
  
  
• Grow at 37°C, shaking until the A550 is between 0.4 and 0.6. To measure this, blank the spectrophotometer with a sample of fresh LB and determine the absorbance of aliquots of culture (maintaining sterility) at half hourly intervals.
  
  
• When the appropriate culture density is reached (this should be within 4 hours), divide into two sterile 50ml universals and chill on ice for 10min and then spin in the Jouan (ie at 4°C) at 2000rpm for 10min. Meanwhile chill sterile 100mM CaCl2 (made fresh from frozen1M stock and milliQ water)
  
  
• Resuspend each bacterial pellet in 15ml of ice cold 100mM CaCl2(ie a total of 30ml for 100ml of culture), with gentle shaking and brief vortexing. Pool the cells. Keep the temperature below 4°C. Leave on ice for 30min.
  
  
• Repeat the centrifugation step and resuspend the pellet, now composed of all of the cultures, in 8.5ml of ice cold 100mM CaCl2.
  
  
• Leave on ice for a further 30min. The cells are now ready to use. Greater competency can be obtained by leaving cells on ice for longer periods- up to 2hours-but this is generally unnecessary for routine subcloning.
  
  
• To store the cells add 1.5ml of ice cold sterile glycerol, mix gently but thoroughly, aliquot into 200µl portions in sterile microtubes and store at -70°C. Test the competency of the cells by transforming a tube with 1ng of plasmid and look for at least 1000 colonies per ng. Transformation procedure involves adding a known amount of plasmid (1-10ng) to freshly thawed competent cells on ice and leaving for 30min. Heat shock for 1.75min in a 42°C waterbath and add 1 ml of LB media. Incubate at 37°C with gentle shaking for 1 hour and plate 10µl on an LB/ampicillen/X-gal/IPTG miniplate (Select plate). Incubate overnight at 37°C and count the colonies. A colony count of 1 to 10million per µg of input plasmid is OK, with no white colonies.

Select plates; 100µg/ml ampicillen in cooled LB agar, pour plate (25ml to 90mm petri dish) and when set spread a mixture of 50µl 20mg/ml X-gal in DMSO and 50µl 1M IPTG in water (filter sterilised).

Note

pBluescript plasmids are not very good at going blue; incubation at 4°C for an hour followed by a couple of hours back at 37°C sometimes inproves colour. If things still really aren’t turning blue then incubation at 42°C can help.