User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/25

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Cutting with HindIII for Cloning

Since I didn't use all of the insert that was cloned using PCR on 6/11/12 and gel cleaned-up on 6/12/12 I can just use restriction enzymes to digest and get more for ligation. The digest is being done for both the wildtype Asc Hb and the M8S/M33S/M103S Asc Hb clones. This will also be done with the midi-prepped pQE-80-L-Kan. For each:

  1. Mix 15μL of "cleaned-up" DNA with 5μL of 10x NEBuffer2, 25μL of sterile dH2O, and 5μL of HindIII enzyme.
  2. Incubate for two hours at 37°C (heat block).
  3. Incubate for 20 minutes at 65°C (water bath).

Cutting with BamHI for Cloning

  1. Mix 45μL of today's HindIII digest products with 5μL of BamHI-HF (HF=high fidelity).
  2. Incubate for two hours at 37°C (heat block).
  3. Add 5μL of 10% SDS solution (for a final concentration of 0.9% SDS).
    • This was done so the enzyme will not remain attached to the DNA during gel electrophoresis.

Running a DNA Gel

  1. A 1.2% agarose gel was made by combining 0.3g agarose with 25mL TAE buffer, microwaving the mixture until boiling, pouring the mixture into gel chamber, and placing a comb for wells.
  2. The first well was loaded with 5μL of pre-mixed ladder.
  3. The second well was loaded with 10μL of the M8S/M33S/M103S/A71M/L40M mixed with 2μL of 6x loading dye.
  4. The first large well was loaded with 50μL of the double digested pQE-80-L-Kan with 10μL of 6x loading dye.
  5. The second large well was loaded with 50μL of the double digested wildtype Asc Hb with 10μL of 6x loading dye.
  6. The third large well was loaded with 50μL of the double digested M8S/M33S/M103S Asc Hb with 10μL of 6x loading dye.
  7. The gel was run at 90V until the dye band had moved about 3/4 of the way down the gel
  8. The gel was then stained in ethidium bromide for about 30 minutes.
  9. The gel was then destained in TAE buffer for about 20 minutes.

Image:Photo_(19).JPG

The bads are light, but since I could see them I could cut them out. It does look like the pQE80-L-Kan has bands at both 3kb and 2kb (and it supposed to about 4.7kb). I will not continue on with this sample since I still have some from the last double digest. Also, it does not look like the PCR worked.

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

The full protocol can be found here.

  1. Three 1.5mL microcentrifuge tubes were weighed and their weights were recorded.
    • Tube for pQE-80-L-Kan double digest: 1.04g
    • Tube for wild-type double digest: 1.04g
    • Tube for triple mutant double digest: 1.05g
  2. The large bands on the gel were cut out of the gel with a razor blade (additional safety required with UV light).
  3. The bands were placed into the microcentrifuge tubes and the tubes were re-weighed.
    • Tube for pQE-80-L-Kan double digest: 1.14g
    • Tube for wild-type double digest: 1.31g
    • Tube for triple mutant double digest: 1.23g
  4. The microcentrifuge tubes will be stored at 4°C overnight.

Split PCR for L40M Mutation

Using a modified version of 2-stage QuikChange the L40M mutation should be inserted into the Colony 1 miniprep product from 6/19/12 (M8S/M33S/M103S/A71M). Trying the last mini-prepped colony from 6/19/12 in the hopes that this will be a successful PCR (because it seems the other attempts from last week were not successful).Hopefully this miniprep has the proper mutation (will be sequenced next week).

Protocol

First Stage

1st stage
Tube sterile H2O Pfu Buffer Col. 1 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 27 μL 5 μL 5 μL 10 μL 0 μL 2 μL 1 μL 50 μL
Experimental-reverse 27 μL 5 μL 5 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

Making Media

  • Make 3 50mL LB broths in bottles (for mini-preps, growing small cultures, etc.): each is 1.25g LB (w/o NaCl) with 0.5g NaCl dissolved in 50mL of dH2O
    • Autoclave all of these broths
    • Also, autoclaved a dry cycle today.
    • Also, autoclaved old and/or contaminated plates today

Transformations

Transformations were done with the M8S/S33L/M103S Asc Hb into NovaBlue and BL21(DE3) cells (lots of different amounts of cells and DNA).

  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 6/21/12: 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, store at 4°C).
  11. Incubate inverted overnight at 37°C.

Start Growths for Minipreps

Colonies of NovaBlue + M103S and NovaBlue + M8S/M33L/M103S that grew on LB/Amp plates many days after the actual transformation, will be grown in 10mL of LB with 100μg/mL of ampicillin at 37°C at 250rpm (one colony per culture, three cultures of each transformation product). If the colonies grow overnight then they will be mini-prepped in the morning.



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