Real-time PCR

Updated 09/26/20

1. Follow these tables to prepare two 3.5X reactions, where you will test two different primer concentrations, each with three different template concentrations. This is done for optimizing the primer and template concentrations and their ratio.
   Don’t add the library (template) to the mix tube.
   For libraries bought from Twist use 2 pg/uL as the 1X. For libraries bought from GenScript (CustomArray), use 2 ng/uL as your 1X.

Reagent	Initial	Final 1X	.1X template	1X template	10X template	3.5X mix
iQ SYBR Green supermix	2x	1x	5 ul	5 ul	5 ul	17.5 ul
Forward	10 uM	0.8 uM	0.8 ul	0.8 ul	0.8 ul	2.8 ul
Reverse (T7)	10 uM	0.8 uM	0.8 ul	0.8 ul	0.8 ul	2.8 ul
Library	X pg/ul	X pg	1 ul of 0.2 pg/ul	1 ul of 2 pg/ul	1 ul of 20 pg/ul
Water (up to 10 ul)			2.4 ul	2.4 ul	2.4 ul	8.4 ul

Reagent	Initial	Final 1X	.1X template	1X template	10X template	3.5X mix
iQ SYBR Green supermix	2x	1x	5 ul	5 ul	5 ul	17.5 ul
Forward	10 uM	0.4 uM	0.4 ul	0.4 ul	0.4 ul	1.4 ul
Reverse (T7)	10 uM	0.4 uM	0.4 ul	0.4 ul	0.4 ul	1.4 ul
Library	X pg/ul	X pg	1 ul of 0.2 pg/ul	1 ul of 2 pg/ul	1 ul of 20 pg/ul
Water (up to 10 ul)			3.2 ul	3.2 ul	3.2 ul	11.2 ul

2. Make a series of titrations for you library (20, 2, 0.2) pg/uL. 10 uL of each is enough.

3. Dispense 9 uL per well (6 in total) in a qPCR plate.

4. Add 1 uL library to each well, according to the table. Pipette well to mix.

5. Cover the plate with an optical adhesive film. Use a comb to press the film onto the plate.

6. Spindown.

7. Place in a real-time PCR, and start the run.