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RBCL PCR USING INDIVIDUAL PCR REAGENTS: TITRATING MGCL2 • • • • PCR is very sensitive and prone to cross-‐contamination. Work as cleanly as possible. Use filtered pipette tips throughout. Always wear disposable gloves (e.g. nitrile gloves). Change gloves frequently. METHOD 1. On ice, thaw primers rbcLa-‐fM13 and rbcLa-‐revM13 (2 µM each), 10X (or 5X) Taq Buffer, 10 mM dNTP Mix and 25mM MgCl2. Once thawed, flick the tubes to mix contents then microfuge briefly. Keep on ice. 2. Remove the DNA polymerase from the freezer and microfuge briefly to settle the contents, and then keep on ice. 3. Label eight 0.5 mL Eppendorf tubes: M1-‐M8. Put in a rack at room temperature. Add the following to the tubes: Reagent Nuclease Free Water (µL) M1 M2 M3 M4 M5 M6 M7 M8 18 16 14 12 10 8 6 4 25 mM MgCl2 (µL) 2 4 6 8 10 12 14 16 Final Mg conc. in the PCR (mM) 0.5 1 1.5 2 2.5 3 3.5 4 4. Cap the tubes. Mix by flicking and microfuge briefly. Then leave at room temperature. 67
