User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/20

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Transformation Results

Nothing grew.

Split PCR for L40M Mutation

Using a modified version of 2-stage QuikChange the L40M mutation should be inserted into the Colony 2 miniprep product from yesterday (M8S/M33S/M103S/A71M). Hopefully this miniprep had the proper mutation (will be sequenced next week).

Protocol

First Stage

1st stage
Tube sterile H2O Pfu Buffer Col. 2 M8S/M33S/M103S/A71M mutated hemoglobin For primer (L40M f) Rev primer (L40M r)dNTPs Pfu Turbo wax
(10X) (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 50 μL
Experimental-reverse 30 μL 5 μL 2 μL 0 μL 10 μL 2 μL 1 μL 50 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
  • 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 Original DNA in PCR Product

This was done with the PCR done today.

  • Remove the wax from the PCR tube.
  • Add 1μL of DpnI to the PCR tube.
  • Incubate the tube on the heatblock at 37°C for one hour.
  • Remove and store at -20°C.

Quantifying DNA

The absorbances of the double-digested pQE80-L-Kan, double-digested wildtype Asc Hb, and double-digested M8S/M33S/M103S Asc Hb were taken at 260 ans 280nm in oreder to calculated the DNA concentration of all of them for better ligations tonight. This procedure was repeated for all three samples:

  1. Using a smaller volume quartz cuvette, 95μL of dH2O was placed in the spectrophotometer and the baseline was corrected with this.
  2. 5μL of DNA was added to the 95μL of water. This was pipetted up and down with a pipette to mix.
  3. The absorbance was taken at 260 and 280nm.

Results

Note: the absorbances are for 20x dilute DNA samples

double-digested pQE80-L-Kan

  • A260 = 0.10
  • A280 = 0.004
  • Absorbance Ratio = 2.2955
  • 20x Dilute DNA concentration = 0.5050 μg/mL
  • Non-dilute DNA concentration = 10.1 μg/mL

double-digested wildtype Asc Hb

  • A260 = 0.002
  • A280 = -0.003
  • Absorbance Ratio = -0.4839
  • 20x Dilute DNA concentration = 0.0750 μg/mL
  • Non-dilute DNA concentration = 1.5 μg/mL

double-digested M8S/M33S/M103S Asc Hb

  • A260 = 0.003
  • A280 = -0.002
  • Absorbance Ratio = -1.4444
  • 20x Dilute DNA concentration = 0.1300 μg/mL
  • Non-dilute DNA concentration = 2.6 μg/mL

Transformations

Transformations will be done today with the M103S PCR product (single mutant), and M8S/M33L/M103S PCR Product.

  1. Place plastic culture tubes on ice for about 15 min.
  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 1, 2, or 3uL of DNA to the appropriately labeled culture tube and let sit in ice for 1 minute.
  5. Add cells (30uL, 40uL, or 50uL) 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 on LB/amp plates (prepared fresh today: 6.25g LB (w/o NaCl) + 2.5g NaCl + 5g agar + 250mL dH2O, autoclave liquid cycle, cool to about 60°C, add 250μL 100mg/mL amicillin, pour plate).
  11. Incubate inverted overnight at 37°C.

DNA ligation with T4 DNA Ligase

This will be done with both the wildtype Asc Hb double-digested insert and the M8S/M33S/M103S Asc Hb double-digested insert. The procedure is based off of the protocol on the NEB website. I does not seem this worked last time, so since I quantified the DNA concentration today I will try to have more of a 3:1 molar ratio. I'm going to try two different reactions for each insert (with different amounts of vactor and insert) in the hopes that one will work.

  1. Mix 2μL of 10x T4 DNA Ligase Buffer, 1μL of the double-digested pQE-80-L-Kan vector, 5μL of the insert, 11μL of autoclaved dH2O, and 1μL of T4 DNA Ligase.
  2. Mix 2μL of 10x T4 DNA Ligase Buffer, 2μL of the double-digested pQE-80-L-Kan vector, 10μL of the insert, 5μL of autoclaved dH2O, and 1μL of T4 DNA Ligase.
  3. Place at 16°C overnight.
    • The thermocycler was used to maintain this temperature overnight.



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