PCR Scun2, Scun22 under Lance et al. conditions
- to replicate original (published) conditions
Calculations
- 12.5µl volume
- 10 mM Tris pH 8.4
- 50 mM KCl
- 25.0 µg/ml BSA
- 0.4 µM unlabeled primer
- 0.04 µM tag labeled primer
- 0.36 µM universal dye-labeled primer
- 1.2 mM MgCl2
- 0.8 mM dNTPs
- 0.5 units JumpStart Taq DNA Polymerase (Sigma)
- 20 ng DNA template
- 10 mM Tris pH 8.4 + 50 mM KCl + 1.2 mM MgCl2
- use Buffer B (10 mM Tris pH 8.3 + 50 mM KCl + 1.5 mM MgCl2
- 0.4 µM of each primer
- [math]\displaystyle{ 12.5 \mu L \bullet \tfrac{0.4 pmol}{\mu L} \bullet \tfrac{\mu L}{20 pmol} = 0.25 \mu L }[/math]
- [math]\displaystyle{ 12.5 \mu L \bullet \tfrac{0.8 nmol}{\mu L} \bullet \tfrac{\mu L}{10 nmol} = 1 \mu L }[/math]
- [math]\displaystyle{ 0.5 U \bullet \tfrac{1 \mu L}{1 U} = 0.5 \mu L }[/math]
PCR
- to mimick Lance et al.
- use Scun (extracted 11-12 January) (species primers were developed) and Scoc (target species)
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