Oct 24, 2020

Public workspaceSensitivity test of toehold

This protocol is a draft, published without a DOI.
  • 1Chung Shan Medical University
Cheng-Ruei Yang
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Protocol Citation: iGEM-CSMU, Cheng-Ruei Yang 2020. Sensitivity test of toehold. protocols.io https://protocols.io/view/sensitivity-test-of-toehold-bijykcpw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 14, 2020
Last Modified: October 24, 2020
Protocol Integer ID: 39256
Preparation
Preparation
Sterilize the bench, and put on a labmat
Thaw the reagents including 1. DNase/ RNase free water (store in -20°C) 2. solution A (store in -80°C) 3. solution B (store in -80°C) 4. RNase inhibitor (store in -20°C) 5.toehold switches with invertase DNA (store in 4°C) 6.miRNA as the trigger (store in 4°C) TemperatureOn ice

Protocols
Protocols
2h 30m 20s
2h 30m 20s
Gently shake and spin down the tube before adding (especially solution A and DNA plasmid)

Add below reagents in to PCR tubes in order:
1. ddH2O(till totallyAmount5 µL )
2. solution AAmount2 µL
3. solution BAmount1.5 µL
4. inhibitorAmount0.2 µL
5. toehold switches with invertase DNAAmount50 ng
6. miRNAAmount50 ng orAmount0 ng (depends on whether its control group or test group)

Centrifuge Duration00:00:20 Centrifigation4000 rpm

20s
Place the PCR tubes in the dry bath incubatorTemperature37 °C Duration02:00:00

2h
Put the PCR tubes TemperatureOn ice after 2 hours

Measure the glucose concentrations of each PCR tubes by glucometer
Put the PCR tubes in the dry bath incubatorTemperature55 °C Duration00:30:00

30m
Measure the glucose concentrations of each PCR tubes by glucometer again