Transformation Experiment Success – Important Tips for Teacher

•  The calcium chloride is supplied in the 15 mL Falcon tubes – it needs to be cold – keep it refrigerated till use, and on ice when directions say ICE.
• Keep plasmids in refrigerator till ready to use.
• Turn on incubator day before use and check to be sure temperature is at 37 degrees C.
• Plate of bacteria needs to be “fresh” – we inoculate and grow up overnight and send to you – you should not refrigerate the plate before use!.
  --If you have been supplied with newly streaked plates and no visible growth, allow to grow overnight in 37 degree C. incubator OR allow two days growth at room temperature.  Check your incubator temperature – if over 37 degrees, then will not work. 
  --IF you have been supplied with fresh plates with visible growth, use within 36 hours OR ELSE if you must wait longer to use, then take fresh LB plate and make transfer of E.coli from your stock plate onto fresh plate – use freshly grown plate for experiment.

• When scooping up bacteria from plate, get a nice visible gob (but not a gob of agar) and then make sure the bacteria are dispersed well into solution – you want to split up the bacterial clumps so that the plasmid will have lots of cells to choose from to enter – only a small percentage of bacterial cells will take up plasmid, so good dispersion maximizes the chances that plasmid finds and gets into a receptive “competent” cell.
•  The Heat Shock Step is very important – take the iced tubes over to 42 degree C water bath, immerse 90 seconds and return to ice.
• Longer recovery time after heat shock gives more transformants but 5 minutes should give results too.