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