User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/08/03

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Amplification of Previous Ligation Products

This procedure was done for the wild-type Asc Hb (Hb pET 3d A) and the triple mutant Asc Hb (M8S/M33S/M103S) that were ligated with pQE-80-L-Kan on 7/11/12, 7/10/12, 7/4/12, and 6/26/12. For the triple mutant ligations the M33S primer was used for amplification and for the wild-type ligations 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.

Running a DNA Gel of Amplified Ligation Products

The ligation products were run on a 1.2% agarose gel at 100V. The gel loading order was: ladder, skip lane, 7/11 wt, 7/11 3x, 7/10 wt, 7/10 3x, 7/4 wt, 7/4 3x, 6/26 wt, 6/26 3x (wt=wildtype ligation with pQE-80-L-Kan, 3x=M8S/M33S/M103S ligation with pQE-80-L-Kan)

Image:Photo_(35).JPG

More DNA there then there was before. The ligation product should be just over 5000 bases, but most of these smudges seem to be smaller (although it's really hard to tell). Maybe I can try to transform with the 7/10 wildtype and 7/4 wildtype ligations to see how that goes (and maybe try others just to see if they transform since everything looks smudgy).



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