Editing 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

<span>100 mM Tris-HCl, 500 mM KCl, and 15 mM MgCl<sub>2</sub>, pH 8.3 @ 25°C.</span>

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

ultrapure water to 50 ml

<span>'''2'''. 50 ml of 150 mM MgCl<sub>2</sub> · 6H<sub>2</sub>O: </span><span>100X</span>

<span>MW 203.3</span>

<span>&nbsp;1.53 g, add ultrapure water to 50 ml</span>

<span>'''3. 50 ml of 250 mM MgCl<sub>2</sub> · 6H<sub>2</sub>O''': </span><span>100X</span>

<span>MW 203.3,</span>

<span>&nbsp;2.54 g, add ultrapure water to 50 ml</span>

'''<span>4. 1% Tween 20:&nbsp;</span>'''<span>100X</span>

<span>5 g in 50 ml ultrapure water.</span>

'''<span>5.&nbsp; 1 M Trehalose MW 342.29 5X</span>'''

<span>17 g in 50 ml ultrapure water.</span>

'''<span>6. </span>10 M 1,2-Propanediol , 10X'''

<span>MW: 76.09, </span>density: 1.036&nbsp;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'''

<span>MW: 132.14</span>,

0.05 * <span>132.14 *2=13.2 g in 50 ml ultrapure water</span>





<span>'''To prepare 1 ml of 2 X Buffer without&nbsp;'''</span>'''1,2-Propanediol'''<span>''':'''</span>

*<span>200 ul </span>750mM Tris-HCl, pH 8.80 (10X)
*20 ul of 2 M (NH4)2SO4 (100X)
*<span>20 ul of 1% Tween 20 (100X)</span>
*<span>20 ul of 250 mM MgCl<sub>2</sub> · 6H<sub>2</sub>O (100X)</span>
*<span>40 ul of 10mM dNTP (50X)</span>
*<span>400 ul of 1 M Trehalose&nbsp; (5X)</span>
*Taq DNA Pol: 25 U or 50 U or 100 U
*Ultrapure Water to 1 ml



<span>'''To prepare 1 ml of 2 X Buffer with'''</span>'''<span style="color:#FF0000">1.5 mM MgCl2 final conc</span> '''<span>'''without&nbsp;'''</span>'''1,2-Propanediol'''<span>''':'''</span>

*<span>200 ul </span>750mM Tris-HCl, pH 8.80 (10X)
*20 ul of 2 M (NH4)2SO4 (100X)
*<span>20 ul of 1% Tween 20 (100X)</span>
*<span>20 ul of 150 mM MgCl<sub>2</sub> · 6H<sub>2</sub>O (100X)</span>
*<span>40 ul of 10mM dNTP (50X)</span>
*<span>400 ul of 1 M Trehalose&nbsp; (5X)</span>
*Taq DNA Pol: 25 U or 50 U or 75 U or 100 U
*Ultrapure Water to 1 ml

<span>'''To prepare 1 ml of 2 X Buffer with'''</span>'''<span style="color:#FF0000">1.5 mM MgCl2 final conc</span> '''<span>'''with'''</span>'''Bromophenol Blue'''<span>''':'''</span>

*<span>200 ul </span>750mM Tris-HCl, pH 8.80 (10X)
*20 ul of 2 M (NH4)2SO4 (100X)
*<span>20 ul of 1% Tween 20 (100X)</span>
*<span>20 ul of 150 mM MgCl<sub>2</sub> · 6H<sub>2</sub>O (100X)</span>
*<span>40 ul of 10mM dNTP (50X)</span>
*<span>400 ul of 1 M Trehalose&nbsp; (5X)</span>
*Taq DNA Pol: 200 U
*20 ul of 1% Bromophenol Blue(100 X)
*200 ul of 50% glycerol(10X)
*Ultrapure Water to 1 ml



<span>'''To prepare 100 ml of 2 X Buffer with'''</span>'''<span style="color:#FF0000">1.5 mM MgCl2 final conc</span> '''<span>'''without&nbsp;'''</span>'''1,2-Propanediol'''<span>''':'''</span>

*2 ml of 2 M (NH4)2SO4 (100X)
*<span>20 ml </span>750mM Tris-HCl, pH 8.80 (10X)
*<span>2 ml of 1% Tween 20 (100X)</span>
*<span>2 ml of 150 mM MgCl<sub>2</sub> · 6H<sub>2</sub>O (100X)</span>
*<span>0.4 ml of 100mM dNTP (500X)</span>
*<span>40 ml of 1 M Trehalose&nbsp; (5X)</span>
*Taq DNA Pol
*Ultrapure Water to 100 ml

<span><span>'''To prepare 1ml of 2 X Buffer with'''</span>'''&nbsp; 1,2-Propanediol'''<span>''':'''</span></span>

*<span>200 ul </span>750mM Tris-HCl, pH 8.80 (10X)
*20 ul of 2 M (NH4)2SO4 (100X)
*<span>20 ul of1% Tween 20 </span>'''<span>(</span>'''<span>100X)</span>
*<span>20 ul of 250 mM MgCl<sub>2</sub> · 6H<sub>2</sub>O </span><span>100X</span>
*<span>40 ul of 10mM dNTP (50X)</span>
*<span>400 ul of 1 M Trehalose&nbsp; (5X</span>)
*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%&nbsp; Tomah E-18-15 will stabilize enzyme GoTaq (US6242235)