User:Tara K. Luckau/Notebook/Team ConGen/2011/01/13

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PCR Scun2, Scun22 under Lance et al. conditions

  • to replicate original (published) conditions

Calculations

  • Lance et al. 2009
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
  • Primer
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]
  • dNTPs
[math]\displaystyle{ 12.5 \mu L \bullet \tfrac{0.8 nmol}{\mu L} \bullet \tfrac{\mu L}{10 nmol} = 1 \mu L }[/math]
  • Taq
[math]\displaystyle{ 0.5 U \bullet \tfrac{1 \mu L}{1 U} = 0.5 \mu L }[/math]


PCR Touchdown

  • to mimick Lance et al.
  • use Scun (extracted 11-12 January) (species primers were developed) and Scoc (target species)



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