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Assignments Due

• Go to the Presentation Schedule
  • Presenters see the guidelines for Journal Club Presentation
  • Everyone else read listed articles and be prepared for discussion

Overview of Experiment

In today's lab, you analyze the assembly reaction on an agarose gel, transform bacteria with assembled vector, and learn how to run a fluorescent plate reader.

Agarose gel analysis of assembly reaction

1. Prepare a mini 1% agarose gel with one 8-well comb (use ~40 ml molten agarose) as on Day 2
2. Add 3 µl 6X LB to 15 µl ITA reaction
3. Load 10 µl NEB Quick-Load 1 kb DNA Ladder
4. Run the gel at 130 V for at least 30 minutes

Transformation of assembled vector

1. Thaw a tube of cells (0.05 ml) on ice for 10 min
2. Add 5 µl of assembled DNA to cells and gently flick the tube 4-5 times to mix DNA and cells
   
   You must be extremely gentle when working with competent cells. These cells are highly sensitive to temperature changes and/or mechanical lysis. Mix cells by gently tapping the tube or swirling with a pipet tip, not by pipetting up & down or vortexing.
   
   
3. Incubate on ice for 30 min
4. Heat shock at 42°C for exactly 10 sec
5. Place on ice for 5 min
6. Add 950 µl of room temperature SOC medium to the mixture
7. Incubate the sample at 37°C for 1 hour with shaking at 250 rpm
8. Mix cells by flicking tube and inverting
9. Pipet 100 µl of transformed cells on a prewarmed LB plate with Amp (50 µg/ml) and Kan (20 µg/ml)
10. Pour 10 - 20 sterile solid glass beads onto the plate, set the plate on the benchtop, and "shake" plate back and forth on the bench top; invert plate to pour off beads (collect in a large beaker -- these can be reused)
11. Pipet 200 µl of transformed cells on a prewarmed LB plate with Amp (50 µg/ml) and Kan (20 µg/ml)
12. Repeat step 10.
13. Let the plates sit 5 minutes at room temperature so that the liquid absorbs into the agar
14. Incubate the plates (upside down) overnight at 37°C
    

Fluorescent plate reader training (Keck 201)

1. plate 100 µl of each dilution in triplicate
2. plate 100 µl PBS in triplicate as a control
   
   
   
   

   YOU WILL HAVE TO COME IN THE DAY BEFORE LAB DAY 6 AND PULL COLONIES (pTetR-GFP).
   The colonies will be grown overnight in 3 ml LB-Amp (50 µg/ml) at 37°C with shaking (225 rpm).

   
   
   1. With your Sharpie, circle 2 well-isolated colonies on the plates.
   2. Put 3 ml of LB-Amp in 2 sterile plastic tubes.
   3. Gently swab a circled colony with a sterile pipet tip. Do not touch any other colonies.
   4. Eject the tip into the appropriately labeled tube.
   5. Slightly loosen the lids and put the tubes in the 37°C shaker overnight.
   6. Store the plates at 4°C.

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   Copyright, Acknowledgements, and Intended Use
   Created by B. Beason (bbeason@rice.edu), Rice University, 21 November 2007
   Updated 2 November 2011