User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/08/16

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Cutting with BamHI for Cloning

  1. Mix 45μL of this morning'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 0.91% SDS).
    • This was done so the enzyme will not remain attached to the DNA during gel electrophoresis.

Running an Analytical/Gel Purification DNA Gel of the pQE-80-L-Kan HindIII/BamHI Product from This Morning

  1. A 1.2% agarose gel was made by mixing 0.3g of agarose with 25mL TAE buffer. This mixture was heated in the microwave for 37 seconds and then poured into a gel chamber with a comb inserted.
  2. When the gel had hardened the comb was removed and 5μL of pre-mixed DNA ladder was loaded into the first lane.
  3. 5μL of the midiprepped pQE-80-L-Kan with 1μL of 6x loading dye was loaded into the third lane.
  4. 5μL of the HindIII digested pQE-80-L-Kan with 1μL of 6x loading dye was loaded into the fifth lane.
  5. 50μL of the double digested pQE-80-L-Kan from this morning with 10μL of 6x loading dye was loaded into the wide lane.
  6. The gel was run at 100V until the dye band had moved about 3/4 of the way down the gel.
  7. The gel was then stained in ethidium bromide for about 30 minutes.

  • Only the brighter, larger band of the double-digested pQE-80-L-Kan will be cut-out for gel purification.

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

  1. The protocol for the Wizard® SV Gel and PCR Clean-Up System was 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).

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, 7μL of the double-digested pQE-80-L-Kan vector, 10μL of the insert, and 1μL of T4 DNA Ligase.
  2. Place at 16°C overnight.
    • The thermocycler was used to maintain this temperature overnight.

The procedure will also be done with both the wildtype Asc Hb double-digested insert and the M8S/M33S/M103S Asc Hb double-digested insert in a NEBuffer and ATP in case there is something wrong with the T4 buffer:

  1. Mix 2μL of 10x NEBuffer 4, 5μL of the double-digested pQE-80-L-Kan vector, 10μL of the double-digested insert, 2μL 10mM ATP, and 1μL of T4 DNA Ligase.
  2. Place at 16°C overnight.
    • The thermocycler was used to maintain this temperature overnight.


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