User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/05/23

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Continuing Expressions

  1. Centrifuge the cultures grown overnight at 3500rpm at 4°C for 15 minutes.
  2. Resuspend each of the pellets in 1mL of LB broth.
  3. Add each of the resuspended pellets with broth to one of the four 2800mL flasks made and autoclaved yesterday with 1L of sterile LB.
  4. Add 1mL 100mg/mL ampicillin to each flask.
    • 100mg/mL ampicillin prepared with 5mL of distilled water and 0.5g of ampicillin, mixed and sterile filtered.
  5. Incubate these flasks at 37°C at 165rpm until OD600=1.2.
  • Actual absorbance at 600nm when induced was 1.4. It took about three hours to reach this absorbance.
  1. Add 1mL mixture of 0.1M IPTG, 1M FeCl3, 0.21M alpha aminolevulinic acid to each flask. Continue to incubate at 37°C overnight.
    • Mixture prepared: 1.5g FeCl3 in 9mL dH2O, let cool. Add 0.22g IPTG and 0.25 alpha aminolevulinic acid to the solution.

Running a DNA Gel of Yesterday's PCR Product

  1. Make a 1.2% agarose gel (0.3g agarose + 25mL TAE buffer, microwave until boiling, then pour into gel chamber, and place comb for wells)
  2. When the gel has solidified, add TAE buffer to the chamber until the gel is completely submerged in buffer
  3. Load 10μL of DNA ladder into the 1st well
  4. Load 10μL of the experimental PCR product from yesterday with 2μL 6x loading buffer into the 3rd well (mix before pipetting into well)
  5. Load 10μL of the control PCR product from yesterday with 2μL 6x loading buffer into the 5th well (mix before pipetting into well)
  6. Run the gel at 100V until the "dye line" has moved about 3/4 of the way down the gel
  7. Place the gel in Ethidium Bromide Stain for about 30 minutes
  8. Move the gel to TAE buffer to destain for about 20 minutes
  9. View under a UV light
    • Be careful when working with ethidium bromide and UV light.

There is a strong band in lane 3, and not in lane 5, which means the PCR worked. A transformation will be tried once it is figured out what is wrong with the cells. Also, there appears to be something that was dyed with ethidium bromide in lane 2, but I think that was just contamination of the gel. Nothing was intended to be in lane 2.



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