ABI StepOne Plus

 

ABI stepone plus

Software: StepOne™ and StepOnePlus™ Software v2.3

See link to Readme file, StepOne™ Software v2.3 Release Notes.

A. General protocol for Sybr-Green-based qPCR

example, kit by QuantaBio qScript One-Step SYBR Green RT-qPCR, w/ ROX.

draft

Simplified StepOnePlus setup for a 96-well plate

StepOne Plus software, do set these options:

Experiment type: Comparative Ct (or Standard Curve if that is your design)
Chemistry: SYBR Green
Passive reference: ROX
Reaction volume: 20 µL
Plate type: 96-well

Per-well reaction mix, 20 µL

For each well:

10.0 µL 2X qScript One-Step SYBR Green Master Mix
0.4 µL qScript One-Step RT
0.4 µL Forward primer (10 µM)
0.4 µL Reverse primer (10 µM)
2.0 µL RNA template
6.8 µL nuclease-free water

Thermal cycling program

Program the run as:

1. 50°C for 10 min
Reverse transcription

2. 95°C for 5 min
Initial denaturation / enzyme activation

3. 40 cycles of:

95°C for 10 sec
60°C for 30 sec → collect fluorescence here

4. Melt curve / dissociation stage: ON

Plate setup suggestions

For a simple full plate:

samples in technical triplicate
include NTC wells for each primer pair
include minus-RT controls if checking for genomic DNA contamination
keep each primer pair grouped together on the plate

Practical reminder

Because this is a ROX kit, do not leave passive reference set to none or a low-ROX option. Set it explicitly to ROX.

Quick bench version

Per well:

10.0 master mix
0.4 RT
0.4 F primer
0.4 R primer
2.0 RNA
6.8 water

Program:

50°C 10 min
95°C 5 min
40 cycles:

95°C 10 sec
60°C 30 sec, read
melt curve on

B. Setting up High Resolution Melting Analysis (melt curve) on Step One Software

Start StepOnePlus software (shortcut on desktop), submit Username: [default ]

From the Menu, choose Advanced Set Up

  1. Experiment Properties

    1. Experiment Name, ex: HRM_PLATE001_

      1. Save to appropriate folder on computer

    2. Instrument: Stepone Plus (96 Wells)

    3. Type of Experiment: Melt Curve

    4. Reagents: SYBR

  2. Plate Setup

    1. Define Target ( Refers to Locus) Add/ Name target as needed

    2. Define Samples (Sample refers to DNA, PCR products by number)

    3. Assign targets and samples tab

      1. Click on wells, click on checkbox by target (assign) and sample (assign) for each well used

  3. RUN Method

    1. Select reaction volume

    2. Melt Curve Stage

    3. ONLY CHANGE drop step 2 temp from 60° to 50° C

      1. Step 1: 100% ramp up 95°, 15 seconds

      2. Step 2: 100% ramp down 50°C, 1 minute

      3. Step 3: to 3% °C ramp up 95°C, 15 seconds

  4. Click Start Run

  5. Click on Analyze button

  6. At run end export file to XLS format, save to appropriate folder on computer

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