Team:iTesla-SoundBio/PCRandPCRPurification

PCR (with NEB Phusion Polymerase)

	Volume (50 uL rxn)	Final Concentration
RNase-free Water	38 uL
5X Phusion Buffer HF	5 uL	1X
10 uM Forward Primer	2.5 uL	0.5 uM
10 uM Reverse Primer	2.5 uL	0.5 uM
Template DNA	0.5 uL	Should be <250 ng
10 mM dNTPs Mix	1 uL	200 uM
Phusion DNA Polymerase	0.5 uL	1.0 unit

PCR Purification (with QIAquick Kit from Qiagen)

1. Add 5 volumes of Buffer PB to the sample and pipette up and down to mix.
2. Transfer the mixture to a spin column and centrifuge for 1 min at 17,900 rcf. Discard flow-through.
3. Add 750 uL of Buffer PE to the column and centrifuge for 1 min at 17,900 rcf. Discard flow-through.
4. Centrifuge for another 1 min to remove residual ethanol.
5. Transfer column to a clean 1.5 mL microcentrifuge tube
6. Add 30 uL of Elution Buffer onto the center of the column membrane and let sit for 5 min.
7. Centrifuge for 2 min at 17,900 rcf.
8. Check the concentration of the sample with Nanodrop.