Guidelines For Preparing The Pcr Mix; Amount Of Cdna In The Pcr Mix; Preparing The Sample-Specific Pcr Mix - Applied Biosystems 7900HT Getting Started Manual

Guidelines for
Preparing the
PCR Mix
Amount of cDNA
in the PCR Mix
Preparing the
Sample-Specific
PCR Mix
Applied Biosystems Micro Fluidic Card Getting Started Guide
Follow these guidelines to ensure optimal PCR performance.
• Do not remove a Micro Fluidic Card from its packaging until the
packaging has reached room temperature and you are ready to
load it with sample-specific PCR mix. Prolonged exposure to
indoor lighting can photo-degrade the fluorescent probes
contained within the Micro Fluidic Card. Do not expose the
Micro Fluidic Card to sunlight.
• Load each Micro Fluidic Card fill reservoir with sample-
specific PCR mix made from a single cDNA sample.
• Load 100 µL of the sample-specific PCR mix per fill reservoir
to ensure adequate filling. Smaller volumes will result in
insufficiently filled Micro Fluidic Cards.
• Do not add additional sample to partially filled Micro Fluidic
Cards. When you centrifuge the card, the sample-specific PCR
mix resuspends the dried TaqMan
the wells of the Micro Fluidic Card. Adding sample after
centrifuging disrupts the resuspended assay positions.
Each 100-µL PCR mix should contain 1 ng to 100 ng of total RNA
converted to cDNA. The cDNA sample volume, with added water or
Tris-EDTA buffer, should be 50 µL.
1 ng to 100 ng is the recommended range. Micro Fluidic Cards
Note:
can accept between 0.1 ng to 1 µg of total RNA converted to cDNA
per port. At less than 1 ng per fill reservoir, however, rare messages
can be difficult to detect.
Load only one sample-specific PCR mix (cDNA
IMPORTANT!
sample + RNase/DNase-free water + TaqMan
Master Mix) per fill reservoir.
CHEMICAL HAZARD. TaqMan Universal
PCR Master Mix 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.
June 23, 2003 9:39 am, MFC_QSG_text.fm
Preparing and Running the Micro Fluidic Cards (PCR)
®
DRAFT
probes and primers within
®
Universal PCR
17