30 June 2009

From 2009.igem.org

Results of 29 June 2009

Result Table
NumberPart NameContent(length/bp)Backbone(length/bp)LocationPlate Result
18R00405TetR_Promoter(???)pSB1A2(2079))P1_06I√(only 2 colonies)
19Q04400RBS+TetR_Coding+T+TetR_Promoter(???)pSB1K3(???)P1_16PX

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.
  2. Add 5µL of DNA in TE to 50µL of competent cells (TOP10)
  3. Allow the DNA and competent cells to sit on ice for 30 minutes
  4. Heat shock at 42ºC for 60 sec in water bath.
  5. Recover on ice for 5 min.
  6. Add 300 µL NZY medium.
  7. Incubate at 37ºC for 2 hr while the tubes are rotating.
  8. Centrifuge and leave about 250 µL liquid; hence, resuspend the E.coli suspension.
  9. Plate 100µL on an LB plate with the appropriate antibiotic.
  • Incubation:
  1. Pick new colonies from plate 9 (FGHIJKLMNO), plate 16(ABCD) & 18(AB). <Since Tube 16AB showed no growth after about 7 hrs incubation, another two colonies(CD) were picked at 17:00>
  2. Incubate with shaking overnight.