Wednesday July 7, 2010

Raida

• Today I completed the transformation of my construct (R0040-I3504) 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

Chris

Today, I drafted the potential budget for this year's project. The total expenses this year are projected to amount to $64 500 with that money being used towards paying students, travelling and lodging at MIT, paying for laboratory materials, and buying T-shirts as well as a mascot suit. Our current financial status is sufficient to cover approximately 80% of this with $54 600 in total funds. These have been provided by BioAlberta, Alberta Innovates Technology Funds, O'Brien Student Centre, and Alberta Heritage Foundation for Medical Research.

As well, in the morning, Dev and I used the Sigma Aldrich GenElute Plasmid Preparation kit and modified instructions to isolate the plasmids of the constructions of I0500 - B0034 and E0040-B0015 as well as the parts of I0500, I13507, and E1010. The procedure used was as follows:

1. Pellet the cells from the overnight cultures using a centrifuge for 20 minutes at a speed of 4000 rpm at 4°C.
2. Discard the supernatant, while being careful not to remove any of the pellet
3. Resuspend the pellet in 200 μL of Resuspension Solution (with RNase A added)
4. Transfer the solution from a Falcon tube to a properly labelled 1.5 μL microcentrifuge tube.
5. Add 200 μL of the Lysis solution and invert gently to mix. Allow the mixture to clear for 4.5 minutes.
6. Add 350 μL of Neutralization Solution and invert the tube 4-6 times to mix.
7. Pellet the microcentrifuge tubes at 14 000 rpm using a microcentrifuge for 15 minutes. The solution will be known as the lysate.
8. Add 500 μL of the Column Preparation Solution to a binding column inside a collection tube. Centrifuge this tube for 1 minute at 14 000 rpm and discard the resulting liquid under the binding tube.
9. Transfer the lysate from the microcentrifuge tube into the binding column while being careful not to transfer any solid. The microcentrifuge tube may now be discarded.
10. Centrifuge the collection tubes at 14 000 rpm for 1 minute and discard the liquid that flowed through the binding column.
11. Add 750 μL of Wash Solution with concentrated ethanol added to the column and centrifuge at 14 000 rpm for 1 minute. Discard the liquid that flowed through into the collection tube.
12. Repeat this procedure a second time with Wash Solution.
13. Spin the tube for 1 minute at 14 000 rpm to dry the column.
14. Transfer the column into a properly labelled 1.5 μL microcentrifuge tube.
15. Add 50 μL of double-distilled water to the column and spin for 1 minute at 14 000 rpm.
16. Use a spectrophotometer to measure the concentration and purity of the plasmids.

In the afternoon, I contacted a General Manager from New England Biolabs about possible sponsorship and received a positive response. They will send some enzymes to us in the coming week.