Crm at 13:47, 8 July 2019

2019-07-08T13:47:44Z

New page

== NEB Taq Buffer, 10 X ==

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

'''1'''. 100 ml of 1M Tris-HCl, pH 8.30 '''Trizma HCl: '''6.14 g '''Trizma Base: '''7.40 g

ultrapure water to 100 ml

'''2'''. 50 ml of 150 mM MgCl2 · 6H2O: MW 203.3

 1.53 g, add ultrapure water to 50 ml

'''3'''. 1% Tween 20:  5 g in 50 ml ultrapure water.

'''To prepare 50 ml 10 X Buffer without Tween 20:'''

5 ml 1M Tris-HCl, pH 8.30.

5 ml 150 mM MgCl2

0.05 * 0.5 * 74.55=1.86 g KCl.

Ultrapure Water to 50 ml.

'''To prepare 50 ml 10 X Buffer without Tween 20:'''

5 ml 1M Tris-HCl, pH 8.30.

5 ml 150 mM MgCl2

+ 0.05 * 0.5 * 74.55=1.86 g KCl.

+  0.5 ml of 10% Tween 20

ultrapure Water to 50 ml.

'''1 M Trehalose MW 342.29'''

17 g in 50 ml ultrapure water.

== 10X Taq Buffer with KCl ==

100 mM Tris-HCl (pH 8.8 or 8.3 at 25°C), 500 mM KCl, 15 mM MgCl2, 0.8% (v/v) Nonidet P40 or Triton X-100.

== 10X Taq Buffer with (NH4)2SO4 ==

750 mM Tris-HCl (pH 8.8 at 25°C), 15 mM MgCl2, 200 mM (NH4)2SO4, 0.1% (v/v) Tween 20.

2X Taq MasterMix

For StepOne ABI

{| style="width: 500px" cellspacing="1" cellpadding="1" border="1"
|-
| Components 
| ul 
|-
| H2O 
| 15.15 
|-
| Buffer 10 X 
| 2.5 
|-
| MgCl2, 50 mM 
| 1.5 
|-
| dNTPs 10 mM 
| 0.5 
|-
| Primers 5 uM 
| 2 
|-
| BSA 10 mg/ml 
| 0.25 
|-
| Taq  5 U/ul 
| 0.2 
|-
| Sybr (10x) 
| 0.4 
|-
| Rox (5x) 
| 0.5 
|-
| cDNA 
| 2 
|}

Increase SYBR and decrease ROX concentrations for Stratagene Mx3005P and BioRad iCycler

Sigma Jump Start Taq Ready Mix. 5ul SYBR green mix MgCl2 1.2 ul 0.4 ul forward primer (10 pmol) 0.4 ul reverse primer (10 pmol) 1 ul water 2ul cDNA (1/4 diluted cDNA)

For High Resolution Melting: use Biotium EvaGreen  and Promega BRYT Green, becuase SYBR Green  inhibits PCR at higher concentrations. EvaGreen at high enough concentrations to saturate the double strand DNA

== Sigma AccuTaq LA and REDAccuTaq LA Buffer ==

AccuTaq LA: 500 mM Tris-HCl, adjust pH to 9.3 using NH4OH, 150 mM (NH4)2SO4, 25 mM MgCl2, 1% Tween 20

REDAccuTaq LA : 500 mM Tris-HCl, adjust pH to 9.3 using KOH, 150 mM (NH4)2SO4, 25 mM MgCl2, 1% Tween 20
[[Category:Buffer Recipe]]

Taq Buffer - Revision history

Crm at 13:47, 8 July 2019