Team:Calgary/7 July 2010

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Revision as of 20:09, 7 July 2010 by Jdchoo (Talk | contribs)

Wednesday July 7, 2010

Raida:

  • Today I completed the transformation of my construct (R0040-I0504) and plated them in 6 different Ampicilin Resistant plates.

Procedure: Transformation

  • 1. Thaw Competent Cells
  • 2. Add 20 uL DNA
  • 3. Ice for 1/2 hour, Heat Shock 5 minutes at 37 degrees Celcius, Ice 5 min.
  • 4. Recover with 250 uL SOC for 30 minutes
  • 5. Spin down at 14 RPM, Concentrate to approximately 100 uL
  • 6. Plate 25 uL on plate 1,2,3 and Plate 50 uL on plate 4,5,6
  • 7. Leave in Incubator overnight for 20 hours

The plates are labeled as following:

  • P1: R0040-I13504 (construct tube C 6 July) 25 uL
  • P2: R0040-I13504 (construct tube D 6 July) 25 uL
  • P3: R0040-I13504 (construct tube F 6 July) 25 uL
  • P4: R0040-I13504 (construct tube C 6 July) 50 uL
  • P5: R0040-I13504 (construct tube D 6 July) 50 uL
  • P6: R0040-I13504 (construct tube F 6 July) 50 uL

Then I left the plates in the Incubator which is at 37 degrees Celcius. I will leave it there until 10:00 am tomorrow (July 8th), which would let the cells grow for 20 hours.


Jeremy: Today I completed the plasmid switch for I0500 into pSB1AK3, mini prep I0500+B0034 with Sigma-Aldrich and finished a restriction digest of EcoRI and PstI on I0500+B0034

  • At the end of the heat killing process:

5 uL Antartic Phosphotase Buffer 5 uL of Water and 1 uL Antartic Phosphotase It was placed in the water bath at 37°C for half an hour and then I ligated the insert and the vector using 5 uL of Insert and 5 uL of Vector 1 uL Quick Ligase 10 uL 2x Quick Ligase Buffer The construct is left in the fridge overnight and transformation will be done tomorrow.

  • see protocol for Gen-Elute Mini Prep Sigma Aldrich Kit
  • Restriction Digest- This is the I0500+B0034 (same for all 4 colonies)
    • -2 uL DNA
    • -0.5uL EcoRI
    • -0.5uL PstI
    • -3.5uL RE2 Buffer
    • -8.5uL H2O
  • 37 degrees for 1 hour and heat kill 80 degrees for 20 minutes
  • ran on 1% agarose gel at 90V


No notebook page exists for this date. Sorry!