30 June 2009

Results of 29 June 2009 Aim:
 * Picking 2 colonies from plate 16; plate 18 for incubation.
 * Transformation of NO.18 & 19 again.
 * Picking 102 colonies from plate 9(contaminated by fungi) for incubation.

Materials:

•TE Buffer

•Plasmids from Kit Plates 1: R0040; Q04400

•Competent E.coli

•NZY Medium

•Ampicllin, Kanamycin Agar Plates

•Eppendorf (labelled)

Methods:
 * Tranformation:
 * 1) Add 7µL of 50ºC TE buffer into R0040well & Q04400well to make the total volumn upto 10µL.


 * 1) Add 5µL 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 9 (FGHIJKLMNO), plate 16(ABCD) & 18(AB). 
 * 2) Incubate with shaking overnight.