Wittwer Lab PCR Master Mix (5X)

Recipe for 500 Reactions (10 µL volume each)
Component volumes in µL
Target Mix
Final [Mg++]		 PolymeraseDNA Polymerase Technology
Cesium Klentaq AC 500X		 BSASigma B8667-5ml
20 mg/ml		 Tris pH 8.3Sigma T8943
2.0 M		 Mg(Cl)₂Sigma M1028
1.0 M		 dNTPsBioline BIO-39029
25 mM each		 LCGreen+Idaho Tech BCHM-ASY-0006
10X		 WaterSigma W4502-1L
1.0 mM 1063125540500257.5
1.5 mM 10631257.540500255
2.0 mM 10631251040500252.5
2.5 mM 106312512.540500250
3.0 mM 10631251540500247.5
4.0 mM 10631252040500242.5
5X Conc.5X1.25 mg/ml250 mM, pH 8.31000 uM each5X
1X Conc.1X0.250 mg/ml50 mM, pH 8.3200 uM each1X
📍 Water Management: To minimize contanimation, use Sigma W4502-1L water for all steps (Master Mix, TE’ dilution, and PCR make-up volumes).
Aliquot 10 mL in 15 mL tubes Aliquot 1 mL in 1.5 mL tubes Store at 4°C

🧪 2 M Tris, pH 8.3 Stock

1. Remove 6.1 ml from a 100 ml bottle of Water (Sigma W3513).
2. Add 13.54 g Trizma pH 8.3 crystals (Sigma T8943).
3. Shake until dissolved. (Density: 1.0718 g/ml).

🧪 100X TE' Preparation

1. Remove 10 ml from 100 ml bottle of 1 M Tris, pH 8.0 (T2694).
2. Add 10 ml 100X TE (Tris/ EDTA: T9285).
3. Result: 1M Tris / EDTA.

🧪 1X TE' Working Buffer (Tris, EDTA)

• Dilute 100X TE' 100-fold using Sigma W4502 water.
• Formula: 10 ml of 100X TE' + 990 ml Water.
• Use for diluting primers and all nucleic acids.