Background control of Gotcha RCA
Chia-Hsien Shih
Abstract
This protocol aims to verify that GotCha design works as expected.
Steps
Preparation
Add 5µL of functional beads(Gotcha) into eppendorf
Centrifuge for 15000rpm and remove supernatant. Make sure that eppendorf should put on DynaMag when removing supernatant.
Protocol of Control group without miRNA
Add 3µL of 10X phi29 polymerase reaction buffer into eppendorf with functional beads
Add 23.4µL RNase-free water
Add 3µL of 2mM dNTPs
Add 0.6µL of 10U/ul phi29 polymerase
Pipetting to mix well
Incubate for 2h 0m 0s at Room temperature
Add 1.5µL of 20X evagreen dye
Add 1.5µL of 0.5M EDTA buffer to suspend the reaction
Protocol of Control group without phi29 polymerase
Add 3µL of 10X phi29 polymerase reaction buffer into eppendorf with functional beads
Add 21µL RNase-free water
Add 3µL of 2mM dNTPs
Add 3µL of 100nM miRNA
Pipetting to mix well
Add 1.5µL of 20X evagreen dye
Add 1.5µL of 0.5M EDTA buffer to suspend the reaction
Measuring
Load 20µL of reaction solution into 384-well plate
Measure the fluorescence excitation and emission intensity
