Real Time PCR Buffer

Real Time PCR Buffer for using with SGI, SYTO-9, SYTO-13, SYTO-82, EvaGreen, LCGreen or ResoLight. (blood resistant)

75 mM Tris-HCl, pH 8.8, 20 mM (NH4)2SO4 , 0.01% Tween 20, 2.5 mM MgCl2, 200 uM dNTP, 0.2 M Trehalose, 1M 1,2-Propanediol, 25 U/ml Taq DNA Pol, 22 nM anti-Taq mAb

100 mM Tris-HCl, 500 mM KCl, and 15 mM MgCl₂, pH 8.3 @ 25°C.

1. 50 ml of 750mM Tris-HCl, pH 8.80 (10X)
Trizma HCl:  18.45 g in 1 L--> 750 mM;  0.92 g in 50 ml--> 750 mM
Trizma Base:  76.95 g in 1 L-->750 mM; 3.85 g in 50 ml --> 750 mM

ultrapure water to 50 ml

2. 50 ml of 150 mM MgCl₂ · 6H₂O: 100X

MW 203.3

 1.53 g, add ultrapure water to 50 ml

3. 50 ml of 250 mM MgCl₂ · 6H₂O: 100X

MW 203.3,

 2.54 g, add ultrapure water to 50 ml

4. 1% Tween 20: 100X

5 g in 50 ml ultrapure water.

5.  1 M Trehalose MW 342.29 5X

17 g in 50 ml ultrapure water.

6. 10 M 1,2-Propanediol , 10X

MW: 76.09, density: 1.036 g/mL

76.09 g in 1 L--> 1 M, 76.09g (73.45 ml) in 100 ml water-->10 M;

7. 50 ml of 2 M (NH4)2SO4, 100 X

MW: 132.14,

0.05 * 132.14 *2=13.2 g in 50 ml ultrapure water

To prepare 1 ml of 2 X Buffer without 1,2-Propanediol:

• 200 ul 750mM Tris-HCl, pH 8.80 (10X)
• 20 ul of 2 M (NH4)2SO4 (100X)
• 20 ul of 1% Tween 20 (100X)
• 20 ul of 250 mM MgCl₂ · 6H₂O (100X)
• 40 ul of 10mM dNTP (50X)
• 400 ul of 1 M Trehalose  (5X)
• Taq DNA Pol: 25 U or 50 U or 100 U
• Ultrapure Water to 1 ml

To prepare 1 ml of 2 X Buffer with1.5 mM MgCl2 final conc without 1,2-Propanediol:

• 200 ul 750mM Tris-HCl, pH 8.80 (10X)
• 20 ul of 2 M (NH4)2SO4 (100X)
• 20 ul of 1% Tween 20 (100X)
• 20 ul of 150 mM MgCl₂ · 6H₂O (100X)
• 40 ul of 10mM dNTP (50X)
• 400 ul of 1 M Trehalose  (5X)
• Taq DNA Pol: 25 U or 50 U or 75 U or 100 U
• Ultrapure Water to 1 ml

To prepare 1 ml of 2 X Buffer with1.5 mM MgCl2 final conc withBromophenol Blue:

• 200 ul 750mM Tris-HCl, pH 8.80 (10X)
• 20 ul of 2 M (NH4)2SO4 (100X)
• 20 ul of 1% Tween 20 (100X)
• 20 ul of 150 mM MgCl₂ · 6H₂O (100X)
• 40 ul of 10mM dNTP (50X)
• 400 ul of 1 M Trehalose  (5X)
• Taq DNA Pol: 200 U
• 20 ul of 1% Bromophenol Blue(100 X)
• 200 ul of 50% glycerol(10X)
• Ultrapure Water to 1 ml

To prepare 100 ml of 2 X Buffer with1.5 mM MgCl2 final conc without 1,2-Propanediol:

• 2 ml of 2 M (NH4)2SO4 (100X)
• 20 ml 750mM Tris-HCl, pH 8.80 (10X)
• 2 ml of 1% Tween 20 (100X)
• 2 ml of 150 mM MgCl₂ · 6H₂O (100X)
• 0.4 ml of 100mM dNTP (500X)
• 40 ml of 1 M Trehalose  (5X)
• Taq DNA Pol
• Ultrapure Water to 100 ml

To prepare 1ml of 2 X Buffer with  1,2-Propanediol:

• 200 ul 750mM Tris-HCl, pH 8.80 (10X)
• 20 ul of 2 M (NH4)2SO4 (100X)
• 20 ul of1% Tween 20 (100X)
• 20 ul of 250 mM MgCl₂ · 6H₂O 100X
• 40 ul of 10mM dNTP (50X)
• 400 ul of 1 M Trehalose  (5X)
• 200 ul of 10 M 1,2-Propanediol (10X)
• Taq DNA Pol: 25 U or 50 U or 100 U
• Ultrapure Water to 1 ml

Loading dye (Green):

Green orange G (yellow) + Xylene cyanol FF (Blue)

Blue dye migrates at the same rate as 3–5kb DNA fragments

Yellow dye migrates at a rate faster than primers (<50bp)

0.005%  Tomah E-18-15 will stabilize enzyme GoTaq (US6242235)