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. Author manuscript; available in PMC: 2026 Sep 3.
Published in final edited form as: Methods Mol Biol. 2018;1708:473–496. doi: 10.1007/978-1-4939-7481-8_24

Table 2.

Preparation of Real-time MSP Reaction Mix Sufficient for 72 sample wells and U, M and Water Controls

Real-time MSP (STEP 2) Reaction Mix 1× (μL) 110× (μL) Final concentration
Reaction mix 10× MSP buffer 2.00 220.0 1×
25 mM dNTP (Denville Scientific) 0.16 17.6 200 μM
ROX 50× (Life Technologies) 0.24 26.4 300 nM
10 mg/mL tRNA (Roche) 0.10 11.0 50 μg/mL
5 U/μL RAMP Taq (Denville Scientific) 0.20 22.0 1.0 U
Water 0.42 46.2
Reaction mix subtotal μL 3.12 343.2
Primes/probes Internal M primer, 5 μM forward 3.20 352 700 nM
Internal M primer, 5 μM reverse 3.20 352 700 nM
Internal U or primer, 5 μM forward 3.20 352 700 nM
Internal U or primer, 5 μM reverse 3.20 352 700 nM
M probe, 100 μM (FAM/TAMRA) 0.04 4.4 200 nM
U or probe, 100 μM (VIC/TAMRA) 0.04 4.4 200 nM
Primer/probe mix subtotal μL 12.88 1416.8
DNA DNA (diluted 1:10−2 or 1:10−4) 4.00
Total μL 20.0
1 Full plate Calculated amount Reaction mix (Rxn) Primers/Probes
Standard curve (7 dilutions)(3× rxn) = 21× (21×)(3.12) = 66 μL (14×)(12.88) = 180 μL
Samples (82×) + (3× extra) = 85× (85×)(3.12) = 265 μL (85×)(12.88) = 1095 μL
Total Amount needed 106× + extra = 110× 99× + extra = 110×