29 June 2009

Aim:
 * Transformation of new (super)parts
 * Incubation

Materials:

•TE Buffer

•Plasmids from Kit Plates 1: R0040; Q04400

•Competent E.coli

•NZY Medium

•Ampicllin, Kanamycin Agar Plates

•Eppendorf (labelled)

Methods:
 * Tranformation Protocol 2:
 * 1) Using the puch tool, puch out the appropriate DNA. Clean the tool between punches.


 * 1) Soak the spots in 5µL of TE that has been warmed to 50ºC for 20 minutes. At this time take the competent cells from the -80º freezer and start defrosting on ice.


 * 1) Add 2µL of DNA in TE to 50µL of competent cells (TOP10)


 * 1) Allow the DNA and competent cells to sit on ice for 30 minutes


 * 1) Heat shock at 42ºC for 60 sec in water bath.


 * 1) Recover on ice for 5 min.


 * 1) Add 300 µL NZY medium.


 * 1) Incubate at 37ºC for 2 hr while the tubes are rotating.


 * 1) Centrifuge and leave about 250 µL liquid; hence, resuspend the E.coli suspension.


 * 1) Plate 100µL on an LB plate with the appropriate antibiotic.


 * Incubation:


 * 1) Pick new colonies from plate 15 & 16.
 * 2) Incubate with shaking overnight.