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.
ReagentInitialFinal 1X.1X template1X template10X template3.5X mix
iQ SYBR Green supermix2x1x5 ul5 ul5 ul17.5 ul
Forward10 uM0.8 uM0.8 ul0.8 ul0.8 ul2.8 ul
Reverse (T7)10 uM0.8 uM0.8 ul0.8 ul0.8 ul2.8 ul
LibraryX pg/ulX pg1 ul of 0.2 pg/ul1 ul of 2 pg/ul1 ul of 20 pg/ul 
Water (up to 10 ul)  2.4 ul2.4 ul2.4 ul8.4 ul

ReagentInitialFinal 1X.1X template1X template10X template3.5X mix
iQ SYBR Green supermix2x1x5 ul5 ul5 ul17.5 ul
Forward10 uM0.4 uM0.4 ul0.4 ul0.4 ul1.4 ul
Reverse (T7)10 uM0.4 uM0.4 ul0.4 ul0.4 ul1.4 ul
LibraryX pg/ulX pg1 ul of 0.2 pg/ul1 ul of 2 pg/ul1 ul of 20 pg/ul 
Water (up to 10 ul)  3.2 ul3.2 ul3.2 ul11.2 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.

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