Applied Biosystems 7500 - Troubleshooting - ROI Image

Chapter 3 Prepare the Reactions
Prepare the Reaction Mix
Applied Biosystems 7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve
Experiments
50
Notes
CHEMICAL HAZARD . TaqMan
®
2 ✕ Universal PCR Master Mix,
No AmpErase
®
UNG may cause eye and skin irritation. Exposure may cause discomfort if
swallowed or inhaled. Read the MSDS, and follow the handling instructions. Wear
appropriate protective eyewear, clothing, and gloves.
1. Label an appropriately sized tube for the reaction mix: RNase P Reaction Mix.
2. For the RNase P assay, add the required volumes of each component to the tube:
3. Mix the reaction mix by gently pipetting up and down, then cap the tube.
4. Centrifuge the tube briefly to remove air bubbles.
5. Place the reaction mix on ice until you prepare the reaction plate.
Preparation
Guidelines
When you prepare your own standard curve experiment:
• If your experiment includes more than one target assay, prepare the reaction mix for
each target assay separately.
• Include excess volume in your calculations to provide excess volume for the loss
that occurs during reagent transfers. Applied Biosystems recommends an excess
volume of at least 10%.
• Include all required components.
• Prepare the reagents according to the manufacturer’s instructions.
• Keep the assay mix protected from light, in the freezer, until you are ready to use it.
Excessive exposure to light may affect the fluorescent probes.
•Prior to use:
– Mix the master mix thoroughly by swirling the bottle.
– Resuspend the assay mix by vortexing, then centrifuge the tube briefly.
– Thaw any frozen samples by placing them on ice. When thawed, resuspend the
samples by vortexing, then centrifuge the tubes briefly.
For More
Information
For more information on preparing the reaction mix, refer to the protocol appropriate for
the reagents you are using in the PCR reactions:
•TaqMan
®
Gene Expression Assays Protocol
• Custom TaqMan
®
Gene Expression Assays Protocol
Component
Volume for
1 Reaction (µL)
Volume for 24 Reactions Plus
10% Excess (µL)
TaqM an
®
Universal PCR Master Mix (2 ✕ ) or
TaqM an
®
2 ✕ Universal PCR Master Mix, No AmpErase
®
UNG
12.50 330.0
RNase P Assay Mix (20
✕ )1.2533.0
Water 8.75 231.0
Total Reaction Mix Volume 22.50 594.0