PCR reaction using Phusion Blood Direct PCR Kit
Overview
Carefully mix and spin down all tubes before opening to ensure homogeneity and improve recovery. The PCR setup can be performed at room temperature.
Protocol
- Table 1. Pipetting instructions.
Component 20 µl reaction 50 µl reaction Final Conc. H2O add to 20 µl add to 50 µl 2x Phusion® Blood PCR Buffer 10 µl 25µl 1X Primer A x µl x µl 0.5 µM Primer B x µl x µl 0.5 µM Phusion® Blood
DNA Polymerase0.4 µl 1 μl Whole blood 1 μl 2.5 μl (see1.1) Optional components for reaction optimization 50 mM MgCl2 0.6 μl 1.5 μl (see1.3) 50 mM EDTA 0.5-1.0 μl 1.25-2.5 μl (see1.3) DMSO 1.0 μl 2.5 μl 5 % (see1.4) - Table 2. Recommended cycling protocol.
Cycle step 2-step protocol 3-step protocol Cycles Temp. Time Temp. Time Lysis of cells (see2.1) 98°C 5 min 98°C 5 min 1 Denaturation
Annealing (see 2.2)
Extension(see2.3)98°C
--
72°C1 s
--
15-30 s/kb98°C
X°C
72°C1 s
5 s
15-30 s/kb35-40 Final extension
72°C
4°C1 min
hold72°C
4°C1 min
hold1