User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/08/15

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A Note About Last Night's Ligation

There seems to have been a power outage last night so the thermocycler was not on and not at 16°C when I came in this morning. The total ligation time was 15 hours and 55 minutes, but I am not sure how much of that time was spent at 16°C and how much of that was spent at room temperature. I have realized that I don't have enough double-digested pQE-80 to re-do the ligation tonight, but I will begin digesting the plasmid and still amplify and transform last night's product.

Amplification of Last Night's Ligation Product

This procedure was done for the wild-type Asc Hb that was ligated with pQE-80-L-Kan last night. The pQE-80 primer was used:

  1. Mix 29μL of Nuclease-free water, 10μL of 5X Phusion HF Buffer, 1μL of 10mM dNTPs, 2.5μL 10μM forward primer, 2.5μL 10μM reverse primer, and 5.5μL of the ligation product.
  2. Add 0.5μL of Phusion DNA Polymerase, mix the solution, and centrifuge shortly.
  3. Put 50μL of wax on top of the mixture and place on the pre-heated block of the thermocycler.
  4. Start with 30 seconds at 98°C on the thermocycler.
  5. Cycle through the following 40 times:
    • 10 seconds at 98°C
    • 30 seconds at 62°C
    • 2.5 minutes at 72°C
  6. A final extension step of 10 min was done at 72°C.
  7. The thermocycler was then held at 4°C.

Most components for this PCR came from the Phusion® High-Fidelity PCR Kit.

Cutting with HindIII for Cloning

This was done with the pQE-80-L-Kan midiprep from 06/19/12.

  1. For the pQE-80-L-Kan, mix 27μL of DNA with 5μL of 10x NEBuffer4, 13μL of sterile dH2O, and 5μL of HindIII enzyme.
  2. Incubate for two hours at 37°C.
  3. Incubate for 20 minutes at 65°C.
  4. Store at -20°C.

Running an Analytical DNA Gel

  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 5μL of DNA ladder into the 1st well.
  4. Load 5μL of last night's ligation product with 1μL 6x loading buffer into the 3rd well (mix before pipetting into well).
  5. Load 5μL of this morning's amplified ligation product with 1μL 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. View under a UV light.
    • Be careful when working with ethidium bromide and UV light.

Image:Photo_(36).JPG

No bands are seen in the third lane (probably because it's only a very small amount of DNA) but there is a smudge in the fifth lane which indicates something is in it. The transformations being done today will better determine if the ligation worked or not.

Transformations

  • Transformations were done with last night's ligation product, this morning's amplified ligation product, the amplified wildtype Asc Hb ligation from 7/4/12, and the amplified wildtype Asc Hb ligation from 7/10/12.
  • Plates were prepared by combining 12.5g of LB with 10g of Agar in 500mL of dH2O. This mixture was autoclaved on a liquid cycle. 500μL of 100mg/mL ampicillin was added to this when it cooled, and plates were then poured.
  1. Place plastic culture tubes on ice for 15 minutes.
  2. Place DNA for transformation on ice.
  3. After DNA has thawed, place cells on ice. (Note - want to minimize the time that the cells are out of freezer and on ice).
  4. Add 3uL of DNA to the appropriately labeled culture tube and let sit in ice for 1 minute.
  5. Add cells (25uL) to the tube on top of the DNA and gently pipette up and down to mix cells and DNA.
  6. Put tube back in ice for 4 minutes.
  7. Heat shock at 42°C for 80 seconds.
  8. Add 100uL of SOC media.
  9. Shake at 37°C for 1 hour
  10. Plate 50uL of culture media.
  11. Incubate overnight at 37°C.


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