(B) Inversion analysis To screen for inversions, set up the PCR (Fig. 5c) as described below. Note that primers are paired either as Out-Fwd + In-Fwd or Out-Rev + In-Rev. Include a negative control. ComponentAmount (μl)Final concentrationHerculase II PCR buffer, 5×101×dNTP, 100 mM (25 mM each)12 mMOut-Fwd or Out-Rev primer, 10 μM10.2 μMIn-Fwd or In-Rev primer, 10 μM10.2 μMHerculase II fusion polymerase1MgCl2, 25 mM21 mMDNA template12 ng μl−1ddH2O33Total50 95Perform a PCR by using the following cycling conditions: Cycle numberDenatureAnnealExtend195 °C, 2 min2-3195 °C, 20 s60 °C, 20 s72 °C, 30 s3272 °C, 3 min96Run 2–5 μl of PCR product on a 1–2% (wt/vol) agarose gel to check for size of the products in the case of deletions, or for the presence or absence of PCR products in the case of inversions. Although these PCR conditions are designed to work with most primers, some primers may need additional optimization by adjusting the template concentration, MgCl2 concentration and/or the annealing temperature.
