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==Procedures== *Thaw the Qiagen, Taq2X enzyme and primers. *Prepare primer mix gouup ** Primer mix blue: Y-1(122), 21-1(200), 1-1(270), 5-1(413), 22-1(757) ** Primer mix Yellow: 11-1(131), 10-1(214), 15-1(297), 17-1(419), 16-1(806) ** Transfer 10 uL from each primer mix stock solution (100 uM for each primer) and mix together -> 20 uM for each primer (total volume: 50 uL) *Master Mix - x 2 (Primers mix groups are various) **Total reactions: 2 rxns 1 rxn x 2 ----------------------------- RNAse-free H2O 15.0 Primer mix group (20 uM) 5.0 ---- Primer mix group 1 or 2 Template (1 ng/uL) 5.0 Qiagen kit enzyme (2X) 25.0 ----------------------------- 50.0 (uL) 1 rxn x 2 ----------------------------- RNAse-free H2O 15.0 Primer mix group (20 uM) 5.0 ---- Primer mix group 1 or 2 Template (1 ng/uL) 5.0 NEB Taq pol.(2X) 25.0 ----------------------------- 50.0 (uL) **Perform PCR reaction in thermocycler: program GENE52 (Tm52, 30 cycles) *Gel Electrophoresis **1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe -> 4 small tray with 8-well comb **Run at 135 V for 30 min. **Loading: Sample 8 uL + 2 uL loading buffer -> 8 uL. Diluted ladder 8 uL *Repeat the same design to perform PCR on primer set2 (23 reactions)
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