Sample cDNA :  thaw your cDNA on ice ( vortex and quick spin before plating )

    Primer stock (+/-): Add 12.5 µl of (+) and 12.5 µl (-) to 1 mL MilliQ H<sub>2</sub>O

PCR Master Mix (10ul rxn; Triplicate)

A	B
MilliQ H2O	10.20ul
SYBR Green	20ul
primers (2.5 μM each set)	4.80ul
Total master mix	35.00ul

Equipment: ABI 7900 Prism

Place 8-tube PCR strips in PCR tube racks (each single tube runs 1 sample and 1 gene)

Add 5 µl cDNA to the bottom of each tube, use 20 uL pipetor (keep on ice)

Add 25 µl (duplicates) or 35 µl (triplicates) of Sybr Mastermix to the 8-well tube (keep on ice)

Use 200uL multi-channel pipette.

Mix using the multichannel and quick spin.

Dispense 10 µl of cDNA/Mastermix (20uL pipetor) into each well on 384 well plate according to plate layout made in advance (keep plate on ice)

Gently blot top of plate with kimwipe (to keep samples from transferring to other wells)

Place clear Adhesive plate cover over the plate.

use brown ‘helper’ to smooth out

pay attention to edges

work from center of the plate out

Spin plate for 5 min at 3500 rpm (4° C)

During spin: set up ABI SDS program (keep plate in centrifuge until ready to run)

Seal plate with sticky film. Vortex and spin down plate 3500rpm for 5 min at 4C

Open SDS 2.3 program

File -> new

One instrument tab: real time -> Connect to machine -> open/close door

Insert plate, aligning A1 to A1

Close door

On layout tab; highlight unused wells, click “omit wells”

Highlight used wells and click “add detector” for each specific gene

Set to 10uL Rxn VL

Check cycle times and temperatures

Add dissociation stage (SYBR primers only)

A	B	C	D	E	F	G	H
Temp C	50	95	95	60	95	60	95
Time	2:00	10:00	0:15	1:00	0:15	0:15	0:55

Stage C&D 40 time

Run plate

Primer Validation Procedure: Set-up is same as above plus cDNA standard curve for each gene in an extra set of 8-tube PCR strips (see workflow file)