ABI PRISM 7000 Sequence Detection Systems Relative ...
ABI PRISM 7000 Sequence Detection Systems Relative ...
ABI PRISM 7000 Sequence Detection Systems Relative ...
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Chapter 4 Generating Data from RQ Plates<br />
Preparing the PCR Master Mix<br />
Follow these recommended general PCR practices:<br />
• Wear a clean lab coat (not previously worn while handling amplified PCR products<br />
or used during sample preparation) and clean gloves when preparing samples for<br />
PCR amplification.<br />
• Change gloves whenever you suspect that they are contaminated.<br />
• Maintain separate areas, dedicated equipment, and supplies for:<br />
– Sample preparation and PCR setup<br />
– PCR amplification and post-PCR analysis<br />
• Never bring amplified PCR products into the PCR setup area.<br />
• Open and close all sample tubes carefully. Try not to splash or spray PCR samples.<br />
• Quick-spin PCR samples whenever residual sample is present on the inside lid<br />
(such as after dropping a tube or when there is condensation on the tube from<br />
heating or thawing)<br />
• Keep reactions and components capped as much as possible.<br />
• Use aerosol-resistant or positive-displacement pipette tips.<br />
• Clean lab benches and equipment periodically with freshly diluted 10% chlorine<br />
bleach.<br />
Preparing the PCR Master Mix<br />
The second step in the two-step RT-PCR procedure is amplifying the cDNA. TaqMan ®<br />
Universal Master Mix reagents provide a PCR mix that may be used with any<br />
appropriately designed primer and probe to detect any DNA or cDNA sequence.<br />
The TaqMan Universal PCR Master Mix Protocol (PN 4304449) explains how to use the<br />
reagents provided in the kit. The following table lists the universal assay conditions<br />
(volume and final concentration) for using the master mix.<br />
CHEMICAL HAZARD. TaqMan Universal PCR Master Mix<br />
may cause eye and skin irritation. Exposure may cause discomfort if swallowed or<br />
inhaled. Read the MSDS, and follow the handling instructions. Wear appropriate<br />
protective eyewear, clothing, and gloves.<br />
Reaction Component<br />
Volume (µL)<br />
Per Sample<br />
Final Concentration<br />
TaqMan Universal PCR Master Mix (2✕) 25.0 1✕<br />
Forward primer 5.0 50 to 900 nM<br />
Reverse primer 5.0 50 to 900 nM<br />
TaqMan probe 5.0 50 to 250 nM<br />
cDNA sample 5.0 10 to 100 ng<br />
Nuclease-free water 5.0 —<br />
Total 50.0 —<br />
DRAFT<br />
September 26, 2003 3:14 pm, C4_RQPlate.fm<br />
Notes<br />
20 <strong>Relative</strong> Quantification Getting Started Guide for <strong>7000</strong> v1.1