I normally use 20 minutes of cold shock then 1 minute of heat shock at 42 degree celsius and 2 minutes of cold shock again and then I put in 1 ml of LB medium and keep in rotatory shaker for 1 hour,then i pipette one for dilute concentration of 50ul from the soultion ,then I centrifuge at 5000rpm for 2 min and discard 900ul of solution,dissolve the pellet in the rest solution and then plate it as concentrated,but I didnt get any colonies for it so far,should I bring changes in the timing of heat and cold treatments??
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