To access your account, log in or register.	shopping cart | log in

Protocols for Multiplex PCR 5X Master Mix FAQs for Polymerases Technical Reference for Polymerases Enzyme Finder NEBcutter NEBuffer Chart Double Digest Finder Isoschizomers DNA Sequences and Maps REBASE

Home > Products > Polymerases > Master Mixes > Multiplex PCR 5X Master Mix Multiplex PCR 5X Master Mix New Catalog # Size Concentration Price Qty   M0284S 100 reactions (50 μl vol) 5 X $210.00 Prices are in US dollars and valid only for US orders. Download: MSDS PDF Description: Multiplex PCR can simultaneously detect two or more products in a single reaction. There is an increasing demand for multiplex PCR techniques in assays conducted in research laboratories and forensic/diagnostic genotyping assays (1,2). Multiplex PCR can also be used for semi-quantitative gene expression analysis using cDNA templates. The NEB Multiplex PCR 5X Master Mix is an easy-to-use solution featuring high quality recombinant Taq DNA Polymerase. The mix is optimized for high yield and robust performance. Its performance is illustrated in a 15-plex PCR reaction using human genomic DNA (Figure 1) and an 8-plex PCR reaction using cDNA products as templates (Figure 2). The 5X formulation allows maximal input of customer primers, template DNAs and additional components. Figure 1: 15-plex PCR using varying amounts of human genomic DNA. 1X Multiplex PCR 5X Master Mix was used with 0.15 μM of each primer. The cycling conditions were 95°C for 1 minute, 35 cycles of 95°C for 20 seconds, 60°C for 1 minute and 68°C for 2 minutes. Figure 2: 8-plex PCR using cDNA products from 1 ng human spleen total RNA. Cycling conditions were 95°C for 1 minute, 30 cycles of 95°C for 20 seconds, 60°C for 30 seconds and 68°C for 2 minutes. Figure 3: Comparison of PCR product yields between single-plex and multiplex PCR reactions. Figure A shows amplification of human genomic DNA fragments (sizes are listed to the left of the gel). Lane 1 is a 3-plex reaction, Lanes 2-4 are single-plex reactions, and Lanes 5–6 are 2-plex reactions; Lane 7 is the 1 kb DNA Ladder (NEB #N3232). Figure B shows the results of expression analysis of five mRNAs using cDNA templates (first strand synthesis was carried out using the ProtoScript Kit with human spleen total RNA); Lane 1 is a 5-plex PCR from cDNA reactions, while Lanes 2–6 are single-plex PCR reactions; Lane 7 is the 2-Log DNA Ladder (NEB #N3200). Source: An E. coli strain that carries the Taq DNA Polymerase gene from Thermus aquaticus YT-1. Reagents Supplied: Multiplex PCR Master Mix Enzyme Properties Heat Inactivation: No Reaction & Storage Conditions Reaction Conditions: 1X Multiplex PCR Master Mix Incubate at 75°C. 1X Multiplex PCR Master Mix: 20 mM Tris-HCl 50 mM KCl 2.5 mM MgCl2 0.3 mM dNTPs 3.2 % Glycerol 0.08 % NP-40 0.07 % Tween-20 100 units/ml Taq DNA Polymerase 30 mM NH4Cl pH 8.9 @ 25°C Unit Definition: One unit of Taq DNA Polymerase is defined as the amount of enzyme that will incorporate 10 nmol of dNTP into acid-insoluble material in 30 minutes at 75°C. Unit Assay Conditions: 1X ThermoPol Reaction Buffer, 200 µM dNTPs including [3H]-dTTP and 200 µg/ml activated Calf Thymus DNA. Concentration: 5 X Storage Temperature: -20°C Notes General notes: Keep at -20°C for long term storage. Multiplex PCR 5X Master Mix is stable at 4°C for six months or after fifteen freeze-thaw cycles. For daily use, we recommend keeping an aliquot at 4°C. Usage notes: Use high quality primers (desalted or HPLC purified). Accurately quantify and adjust primer concentrations to 50 μM in 0.5X TE Buffer. Individually test the PCR primer pairs, preferentially in a temperature-gradient PCR machine. Mix all primers equally at 1 μM in 0.5X TE buffer. Test multiplex PCR with equal molar concentration of all primer pairs, preferentially in a temperature-gradient PCR machine. Quality Control for Current Lot Quality control values for a specific lot can be found on the datacard which accompanies each product. 15-plex PCR: The Multiplex PCR 5X Master Mix is able to amplify all bands in a 15-plex PCR using 10 ng human genomic DNA in 35 PCR cycles. References Beggs et al. (1997) Nucleic Acids Res., 25, 3957-3958. Krenke et al. (2002) J. Forensic Sci., 47, 773-785. Henke et al. (1997) Nucleic Acids Res., 25, 3957-3958. Sun, Y. et al. (1993) Biotechniques, 15, 372-374. Sarkar, G. et al. (1990) Nucleic Acids Res., 18, 7465.