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
 * $$12.5 \mu L \bullet \tfrac{0.4 pmol}{\mu L} \bullet \tfrac{\mu L}{20 pmol} = 0.25 \mu L$$


 * dNTPs
 * $$12.5 \mu L \bullet \tfrac{0.8 nmol}{\mu L} \bullet \tfrac{\mu L}{10 nmol} = 1 \mu L$$


 * Taq
 * $$0.5 U \bullet \tfrac{1 \mu L}{1 U} = 0.5 \mu L$$

PCR

 * to mimick Lance et al.
 * use Scun (extracted 11-12 January) (species primers were developed) and Scoc (target species)
 * [[Image:20110112_PCROpt_Touchdown.jpg|800 px]]


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