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

Asc Hb Project

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Cutting with BamHI for Cloning Mix 45μL of this morning's HindIII digest product with 5μL of BamHI-HF (HF=high fidelity). Incubate for two hours at 37°C. 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 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. When the gel had hardened the comb was removed and 5μL of pre-mixed DNA ladder was loaded into the first lane. 5μL of the midiprepped pQE-80-L-Kan with 1μL of 6x loading dye was loaded into the third lane. 5μL of the HindIII digested pQE-80-L-Kan with 1μL of 6x loading dye was loaded into the fifth lane. 50μL of the double digested pQE-80-L-Kan from this morning with 10μL of 6x loading dye was loaded into the wide lane. The gel was run at 100V until the dye band had moved about 3/4 of the way down the gel. The gel was then stained in ethidium bromide for about 30 minutes. Error creating thumbnail: Unable to save thumbnail to destination 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 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. 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. 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: 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. Place at 16°C overnight. The thermocycler was used to maintain this temperature overnight.