User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/07

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Split PCR for M103S Mutation

In order to study the individual mutations and their effects on the heme I need to introduce the M103S mutation (methionine to serine at position 103) to the wild-type plasmid (Hb pET 3d A).

Using a modified version of 2-stage QuikChange the M103S mutation should be inserted into the wild-type plasmid.

Protocol

First Stage

1st stage
Tube sterile H2O Pfu Buffer Hb pET 3d A For primer (A71M f) Rev primer (A71M r) dNTPs Pfu Turbo wax
(10X) (12.5 ng/μL) (12.5 ng/μL) (10 mM ea) (2.5 U/μL)
Experimental-forward 27 μL 5 μL 5 μL 10 μL 0 μL 2 μL 1 μL 25 μL
Experimental-reverse 27 μL 5 μL 5 μL 0 μL 10 μL 2 μL 1 μL 25 μL
(-) Control-forward 28 μL 5 μL 5 μL 10 μL 0 μL 2 μL 0 μL 25 μL
(-) Control-reverse 28 μL 5 μL 5 μL 0 μL 10 μL 2 μL 0 μL 25 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

Digestion of Wild-type DNA

  • The wax was removed from the PCR product and the control tube.
  • 1μL of DpnI was added to the PCR product and the control tube and they were put on a heat block at 37°C for one hour.

Spectroscopic Studies of the Triple Mutant

More spectra were taken of Asc Hb M8S/M33S/M103S from 200-800nm today. Spectra were taken of:

  • 25mM Tris, 50mM NaCl, pH 8 (blank)
  • Concentrated Asc Hb M8S/M33S/M103S (1.2mM)
  • 4 times dilute Asc Hb M8S/M33S/M103S (0.3mM)
  • 8 times dilute Asc Hb M8S/M33S/M103S (0.15mM)

The absorbances of the spectra have been corrected with the buffer absorbances and the data has been plotted in Excel:


Attempt to Make and Run a Protein Gel

Since there have been problems with making protein gels lately we borrowed some KB Plus 6.5% GEL MATRIX (used for genotyping) to see if our APS and TEMED could polymerize it. We will also see if we can run it as a protein gel. The protocol for normally using the gel is found here [[1]]. This is how I made and used the gel today:

  1. 10% Ammonium Persulfate (APS) was prepared with 0.1g of APS and 1mL of dH2O.
  2. 10mL of KB Plus 6.5% GEL MATRIX was measured out and then left at room temperature for about a half hour.
  3. 75μL of 10% APS and 7.5μL of TEMED were added to the KB Plus 6.5% GEL MATRIX.
  4. This was poured between a short plate and a spacer plate. A 15-well comb was inserted and the gel was given time to solidify.
  5. Remove the comb and transfer the gel within the plates to the Electrode Assembly with the short plate facing inward (use a buffer dam on the other side).
  6. Fill the Electrophoresis chamber with Tris-glycine electrophoresis buffer (25mM Tris, 250mM glycine, 0.1% SDS)
  7. Load 10μL of pre-mixed SDS-PAGE broad range ladder into the first well.
  8. Load 4μL of concentrated Asc Hb M8S/M33S/M103S mixed with 1μL of 5x loading buffer (pre-mixed) into the wells. Do this for each tube of the concentrated protein (Tubes 1-3) and load the mixtures in lanes 3, 5, and 7.
  9. Run the gel at 200V for about a half hour.
  10. Remove the gel from in-between the glass plates and immerse it in the staining solution (0.25% Coomassie Brilliant Blue R250 (w/v) in 40% (v/v) methanol and 10% (v/v) glacial acetic acid).
  11. Let stain for ~2 hours.
  12. Remove from stain, rinse with destain solution (50% methanol, 10% acetic acid). Immerse the gels in destain solution, microwave for 30 seconds, and let destain overnight.
  • Fresh 5x stock Tris-glycine electrophoresis buffer was made today because the stock we had turned yellow. This buffer was made by dissolving 15.1g of Tris base, 94g of glycine, and 5g of SDS in 1000mL of dH2O.

Transformations

A transformation will be done with the test plasmid that Novagen sends with its competent cells to see if M15MA cells that were made competent yesterday are actually competent. Also, the PCR Product from 5/22/12 (M8S/M33S/M103S/A71M) will be transformed into the new NovaBlue cells that came in a shipment today.

  1. Take sterile microcentrifuge tubes and place them on ice.
  2. Mix 50μL of Competent E.coli with 5μL of either the Novagen Test Plasmid or PCR product in the cold, sterile tubes.
  3. Incubate these mixtures for 30 minutes on ice.
  4. Transfer the tubes to a heat block at 42°C for 90 seconds, and then transfer the tubes back to ice for 5 minutes.
  5. Add 200μL of SOC media to the cells/plasmid or PCR product.
  6. Shake the mixtures at 250rpm at 37°C for 45 minutes.
  7. Spread 100μL of each mixture on an LB plate with 100μg/mL ampicillin.
    • The LB plates were made with 1.75g LB, 1.4g Agar, and 70mL of distilled water. The mixture was autoclaved on a liquid cycle. When the mixture cooled to about 60°C 70μL of 100mg/mL ampicillin was added to it and it was poured into two sterile petri dishes.
  8. Incubate the plates overnight (inverted) at 37°C.




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