User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/06/18

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Gel Purification of Double-Digested pQE-80-L-Kan

  1. Add 5μL of 10% SDS solution (for a final concentration of 0.9% SDS) to the double digested pQE-80-L-Kan that was stored at -20°C over the weekend.
    • This was done so the enzyme will not remain attached to the DNA during gel electrophoresis.
  2. A 1.2% agarose gel was loaded with 50μL of the double digested product with 10μL of 6x loading dye.
  3. The gel was run at 90V until the dye band had moved about 3/4 of the way down the gel
  4. The gel was then stained in ethidium bromide for about 30 minutes.
  5. The gel was then destained in TAE buffer for about 20 minutes.
  6. After the band was cut out of the gel it was stored at 4°C for about an hour.
  7. 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).

Myoglobin Expression

  1. Add approximately 5 colonies of BL21(DE3) + wt swMb pT7-7 to each flask with 50mL LB with 100μg/mL ampicillin.
  2. Incubate at 37°C at 200rpm all day and 250rpm O/N.
    • Note: Growth was started at 11:18am.

Making Media

  • 100mL LB: 2.5g LB (w/o NaCl) + 1g NaCl + 100mL dH2O
  • 50mL LB: 1.25g LB (w/o NaCl) + 0.5g NaCl + 50mL dH2O
    • Made two of these.

All of these were autoclaved on a liquid cycle. dH2O was autoclaved as well.

  • 1L terrific broth: 12g tryptone + 24g yeast extract + 4mL of glycerol, diluted to 900mL
    • Four of these were made.
    • Also, 4 100mL potassium phosphate solutions were made: 2.31g KH2PO4 + 12.54g K2HPO4
    • The potassium phosphate solution will be added to the terrific broth before it is innoculated with cells.

All of these were autoclaved on a liquid cycle.

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.

  1. Mix 2μL of 10x T4 DNA Ligase Buffer, 4μL of the double-digested pQE-80-L-Kan vector, 12μL of the insert, 1μL of autoclaved dH2O, and 1μL of T4 DNA Ligase.
  2. Place at 16°C overnight.
    • The thermocycler was used to maintain this temperature overnight.

Start Growth for Minipreps and Midipreps

All of these cultures were grown overnight at 37°C at 250rpm.