http://www.jove.

com/video/630/pyrosequencing-a-simple-method-for-accurate-genotyping Pyrosequencing Protocol Complete pyrosequencing software setup (user and password=user) 1) Choose new SNP RUN 2) give appropriate name 3) choose cdt003 for the options 4) pick the SNP you are running for each specific well 5) give each well an identity (e.g., BP005, V135, etc) PCR Plate Preparation 1) 5-10ul of each PCRd sample or blank control/well 2) turn on foil heater and PSQ heat block 3) Add 4400ul super H2O + 4400ul binding buffer + 220ul beads (be sure to thoroughly mix beads) to a trough 4)Add 80ul of the mixture in step 3 to each well 5) Cover with a piece of foil (be careful of which side down), apply heat for 3 seconds, press on foil with tissue to ensure adequate adhesion 6) put PCR plate onto shaker and shake for 10 minutes and when ready with PSQ plate go directly to suctioning protocol PSQ Plate Preparation 1) Get white PSQ plate 2) multiply 0.3ul of PSQ primer/well and 10ul of annealing buffer/well by number of wells being PSQd and mix in eppindorf (make a little extra) 3) Add apprx 11ul of the mixture in step 2 to each well of the PSQ plate 4) Place in vacuum holding Position Suctioning Protocol 1) Be sure reservoir for liquid waste has enough room before starting and fill each container with the appropriate chemical. Once both PCR and PSQ plates are prepared and PCR plates have been shaking for 10 minutes you may begin the following step 2

2) Switch on vacuum = both green and metal switch 3) Rotate suctioner in hand 4) Apply vacuum to your PCR plate, allow to completely suction up your PCR liquids in each well, check to be sure you got it all. After suctioning lift up tool and rotate in hand 5) place suctioning tool in ethanol, leave for 5 seconds after seeing liquid being sucked up in tube; lift tool out and rock 6) Do same for NaOH 7) Do same for washing solution 8) Hold tool OVER PSQ plate without actually touching the PSQ plate. 9) Turn off BOTH switches, allow pressure dial to go to zero and no more bubbles being sucked up into tube (you may pull off tube on pump to speed this up) 10) Place tool into PSQ plate and rock gently to ensure mixing 11) Once mixed, place immediately onto heat block for 3 minutes 12) Take off of heat block and cool for 10 minutes 13) Place PSQ plate into pyrosequencer ES ACGT Dispenser Preparation 1) Figure out how much of each ingredient you need from the PSQ program Run section 2) Run water through tips to ensure liquid flow 3)Prepare as needed A, C, G, and T with equal parts 1x TE buffer into each dispensing tip 4) Prepare E and S and needed (dont need equal parts TE buffer) 5) Place tips in dispenser holder, put into pyrosequencer, place test PSQ plate in pyrosequencer and do test dispensation by selecting this option under instrument and manage section of the software 6) If ACGT and ES dots visible then test dispensation success and can do PSQ run. If not, troubleshoot