User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/11

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Diluting Primers for PCR

  • Forward Primer: came with 22.1nMoles, so I added 221μL of sterile dH2O to make the primer 100μM
    • I then took 50μL of the 100μM primer and added 450μL of sterile dH2O to make the final primer 10μM
  • Reverse Primer: came with 27.5nMoles, so I added 275μL of sterile dH2O to make the primer 100μM
    • I then took 50μL of the 100μM primer and added 450μL of sterile dH2O to make the final primer 10μM

PCR for Cloning

This procedure was done for the wild-type Asc Hb (Hb pET 3d A) and the triple mutant Asc Hb (M8S/M33S/M103S):

  1. Mix 34.5μL of Nuclease-free water, 10μL of 5X Phusion HF Buffer, 1μL of 10mM dNTPs, 2.5μL forward primer, 2.5μL reverse primer, and 1μL of either plasmid.
  2. Add 0.5μL of Phusion DNA Polymerase, mix the solution, and centrifuge shortly.
  3. Put 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 30 times:
    • 30 seconds at 98°C
    • 30 seconds at 62°C
    • 15 seconds at 72°C
  6. A final extension step of 5 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 to Extract and Purify PCR Clones

  1. A 1.2% agarose gel was made by mixing 0.3g of agarose with 25mL TAE buffer. This mixture was heated in the microwave for 37 seconds and then poured into a gel chamber with a comb inserted.
  2. When the gel had hardened the comb was removed and 10μL of DNA ladder was loaded into the wide lane.
  3. 25μL of the wild-type PCR product with 5μL of 6x loading dye was loaded into the first large lane.
  4. 25μL of the triple mutant PCR product with 5μL of 6x loading dye was loaded into the second wide lane.
  5. The gel was run at 90V until the dye band had moved about 3/4 of the way down the gel.
  6. The gel was then stained in ethidium bromide for about 30 minutes.
  7. The gel was then destained in TAE buffer for about 20 minutes.

Beginning the The Wizard® SV Gel and PCR Clean-Up System Protocol

The full protocol can be found here.

  1. The gel was viewed a photographed under UV light.
  2. Two 1.5mL microcentrifuge tubes were weighed and their weights were recorded.
    • Tube for wild-type PCR: 1.03g
    • Tube for triple mutant PCR: 1.03g
  3. The large bands on the gel (at ~0.5kb) were cut out of the gel with a new razor blade (additional safety required with UV light).
  4. The bands were placed into the microcentrifuge tubes and the tubes were re-weighed.
    • Tube for wild-type PCR: 1.13g
    • Tube for triple mutant PCR: 1.21g
  5. The microcentrifuge tubes will be stored at 4°C overnight.

Split PCR for S33L Mutation

Now we're going to mutate the Serine (we mutated at the 33rd position from a Methionine) into a Leucine at the 33rd position. The triple mutant Asc Hb plasmid was used (M8S/M33S/M103S). The primer was designed however to change a Methionine in that position to a Leucine.

Protocol

First Stage

1st stage
Tube sterile H2O Pfu Buffer M8S/M33S/M103S mutated hemoglobin Forward primer (M33L f) Reverse primer (M33L r) dNTPs Pfu Turbo wax
(10X) (~100 ng/μL) (12.5 ng/μL) (12.5 ng/μL) (10 mM ea) (2.5 U/μL)
Experimental-forward 30 μL 5 μL 2 μL 10 μL 0 μL 2 μL 1 μL 30 μL
Experimental-reverse 30 μL 5 μL 2 μL 0 μL 10 μL 2 μL 1 μL 30 μL
(-) Control-forward 31 μL 5 μL 2 μL 10 μL 0 μL 2 μL 0 μL 30 μL
(-) Control-reverse 31 μL 5 μL 2 μL 0 μL 10 μL 2 μL 0 μL 30 μL
  • Combine all components of each mixture in sterile PCR tubes, except for Pfu Turbo and wax, mix well, but gently
  • Add Pfu Turbo and mix gently with pipet
  • Add wax on top of mixture and place on thermoclycler
  • 30" @ 95°C
  • Cycle through the following six times:
      1. 30" @ 95°C
      2. 1' @ 55°C
      3. 5' @ 68°C
  • Remove from thermocycler, and pipet wax off of each reaction mixture
  • Begin second stage

Second Stage

2nd stage
Tube 1st stage-forward 1st stage-reverse Pfu Turbo wax
(2.5 U/μL)
Experimental 25 μL 25 μL 1 μL 50 μL
(-) Control 25 μL 25 μL 0 μL 50 μL
  • Combine mixtures from first stage and mix together gently with pipet, then add Pfu Turbo and mix again gently with pipet
  • Add wax on top of mixture and place on thermoclycler
  • 30" @ 95°C
  • Cycle through the following 20 times:
      1. 30" @ 95°C
      2. 1' @ 55°C
      3. 5' @ 68°C
  • 15' @ 68°C
  • hold @ 4°C


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