User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/13

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Cutting with BamHI for Cloning (Double Digest)

  • This will be done with the already HindIII digested DNA from yesterday (both wildtype Asc Hb and the M8S/M33S/M103S Asc Hb clones).
  • What NEB suggests for double digests can be found here: [1]
    • Lucky for me I decided to do it sequentially anyways, and I happened to pick the enzyme with the NEBuffer with the lower salt concentration. The salt concentrations for the two buffers don't seem dramatically different, so I'm going to try it without adding extra salt today.

Today's digest:

  1. Mix 45μL of yesterday's HindIII digest product with 5μL of BamHI-HF (HF=high fidelity).
  2. Incubate for two hours at 37°C.
  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.
  4. A 1.2% agarose gel was loaded with 50μL of the double digested products with 10μL of 6x loading dye.
  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.
  8. The protocol for the Wizard® SV Gel and PCR Clean-Up System was then followed: procedure. The DNA purification was done by centrifugation. The following deviations from the procedure were done:
    • The tabletop centrifuge we have only has a max speed of 13200rpm, so this speed was used instead of 14000rpm (which may affect yield).
    • The step to evaporate the additional ethanol with no cover on the centrifuge was done in a mini centrifuge for 1.5min (only has a max speed of about 6000rpm).

Making Media and Ampicillin for Expression

  • Make 2 1L LB broths: each is 25g LB with 1L of dH2O
  • Make 2 1L LB broths: each is 25g LB (w/o NaCl) + 10g NaCl with 1L of dH2O
  • Make 4 50mL LB broths: each is 1.25g with 50mL of dH2O
    • Autoclave all of these broths
  • Prepare 5mL 100mg/mL Ampicillin
  • 0.5g ampicillin + 5mL distilled H2O
    • Sterile Filter the solution

Transformations

Transformations were done with the M8S/M33S/M103S/A71M Asc Hb into NovaBlue cells (lots of different amounts of cells and DNA). One transformation was also done with wt swMb pT7-7 (myoglobin) into BL21(DE3) cells (2μL DNA, 30μL cells).

  • Plates were prepared by combining 12.5g of LB (w/o NaCl) with 5g of NaCl and 10g of Agar in 500mL of dH2O. This mixture was autoclaved on a liquid cycle. 500μL of 100mg/mL ampicillin was added to this when it cooled, and plates were then poured.


  1. Place plastic culture tubes on ice for 15 minutes.
  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.
  11. Incubate overnight at 37°C.

Start Growth to Make BL21(DE3) Cells Competent

  1. Add 5μL of BL21(DE3) cells to 10mL of LB.
  2. Incubate overnight at 37°C at 250rpm.

Start Growth for Expression of Wildtype Hemoglobin

  1. Add 50μL of 100mg/mL ampicillin to each 50mL LB.
  2. Using a sterile, wooden stick, scrape the BL21(DE3)+ wt Asc Hb glycerol stock and then swirl the stick in the LB/Amp.
  3. Repeat for three other LB cultures.
  4. Incubate these mixtures overnight at 37°C at 250rpm.

See if Transformations from Last Night Really Worked

The colonies on the plates from last night had really small colonies (if they had any colonies at all). I'm testing to see if the colonies from these plates will grow in LB/Amp tonight. I took random colonies from the plates and put them in 3mL of LB with ampicillin at a concentration of 100μg/mL. The cultures will incubate overnight at 37°C at 250rpm.


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