HotStart Taq DNA Polymerase basic kit, 500 U

For detailed product information please refer to the IFU

Product Details

Source:

The Enzyme is purified from an E. coli strain carrying a plasmid with Taq DNA polymerase gene from Thermus aquaticus YT-1. After production, it is chemically modified into a Hot Start enzyme, that requires excessive thermal activation while ensuring high specificity. 

Unit definition:

One unit is defined as the amount of enzyme required to catalyze the incorporation of 10 nmoles of dNTPs into acid insoluble material in 30 minutes at 72^oC.

Reagents supplied:

PD012S kit
Cat No	Description	Tubes No.
PD002-0.100	HotStart Taq DNA polymerase, 5u/μl, 500 U (50 mM Tris-HCl (pH 7.9 @ 25oC), 50 mM KCl, 0.1 mM EDTA, 50% glycerol, 0.5% IGEPAL, 1 mM DTT, 0.5% Tween-20)	1
BR006-1.4	10x Taq DNA polymerase Buffer with 15mM MgCl2 (100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15mM MgCl2)	1
BR007-1.4	10x Taq DNA polymerase Buffer w/o MgCl2 (100 mM Tris-HCl (pH 8.3), 500 mM KCl)	1
BR008-1.4	25mM MgCl2	1
BR028-250	dNTPs mix 10mM each, 0.250 ml	1
BR033-1.8	Nuclease Free Water, 1.8 ml	2

PCR Guidelines:

Reactions may be set at room temperature.

PCR recommended reaction:

Components	25μl assay	Final Concentration/ Quantity (25μl assay)	50μl assay	Final Concentration/ Quantity (50μl assay)
10x Taq pol. Buffer with 15mM MgCl2	2.5μl	1x	5μl	1x
dNTP mix 10mM each	0.5μl	0.2 mM each	1μl	0.2 mM each
10μΜ forward primer	0.5μl	0.2-0.8 μM	1 μl	0.2-0.5 μM
10μΜ reverse primer	0.5μl	0.2-0.8 μM	1 μl	0.2-0.5 μM
Template DNA	Variable	10 fg-500 ng*	Variable	100 fg-500 ng*
Hot Start Taq DNA pol. (5u/μl)	0.25μl	1.25 u/25 μl reaction (0.05 u/μl)	0.25-0.5μl	1.25-2.5 u/50 μl reaction (0.025-0.05 u/μl)
Sterile ultrapure water	Up to 25 μl		Up to 50 μl

PCR recommended conditions:

Step	Temperature	Time
Initial denaturation/Activation	95oC	15 min
25-40 cycles
Denaturation	95oC	30sec
Annealing	45-68oC*	20sec
Extension	72oC	1min/kb
Final extension	72oC	5 min
Hold	4-12oC	Indefinitely

Initial activation for 15 minutes is very important in the case of reaction with very low PCR template copies and for obtaining the maximum efficiency in qPCR setups (either Taqman or Sybr Green chemistry).   DO NOT USE LOWER THAN 10 min!!

Functional Quality Control:

HotStart Taq DNA Polymerase has been tested for performance in the polymerase chain reaction (PCR) using 1.25 units of enzyme to amplify:

1. a 800-bp region of Human genomic DNA using 0.25 ng of E.coli gDNA as substrate in a 25μl reaction.
2. a 5000-bp region of Lambda DNA using 20ng as substrate in a 50μl reaction.

Other Quality Controls:

Certified as DNase-RNase free using fluorescently labeled RNase and DNase probes. DNA free contamination is tested extensively through PCR for the detection of E.coli 16S ribosomal DNA. More details concerning quality controls could be found in respective Certificate of analysis.

Shipping

Shipped on blue ice or dry ice

Storage conditions

Store at -20^οC ± 5^οC

Shelf life:

24 months upon production

Other applications:

1. qPCR on DNA templates

PCR recommended reaction: 

Taq Man qPCR with kit components			Taq man qPCR EQ protocol
Components	Standard 20μl assay	Final Concentration/  Quantity (20 μl assay)	Components	Standard 20μl assay	Final Concentration/  Quantity (20 μl assay)
10x Taq pol. Buffer w/o MgCl2	2μl	1x	5x One Step RT-qPCR buffer* (BR030)	5 μl	1x
25mM MgCl2	2 μl	1.5 -4 mM
dNTP mix 10mM each	0.4μl	0.2 mM each
10μΜ forward primer	0.8μl	0.2-0.8 μM  For single target 0.4 μM for multiplex 0.2 μΜ or lower	10μΜ forward primer	0.8 μl	0.2-0.8 μM  For single target 0.4 μM for multiplex 0.2 μΜ or lower
10μΜ reverse primer	0.8μl	0.2-0.8 μM  For single target 0.4 μM for multiplex 0.2 μΜ or lower	10μΜ reverse primer	0.8 μl	0.2-0.8 μM  For single target 0.4 μM for multiplex 0.2 μΜ or lower
10 μΜ Taqman Probe	0.4 μl	0.2-0.5 μM (~ half or lower than the primer conc.)	10 μΜ Taqman Probe	0.4 μl	0.2-0.5 μM (~ half or lower than the primer conc.)
Template DNA	Variable	10 fg-500 ng	Template DNA	variable	10 fg-500 ng
Hot Start Taq DNA pol. (5u/μl)	0.25-0.5 μl	1.25-2.5 u/20 μl reaction  (0.0625-0.125 u/μl)	Hot Start Taq DNA pol. (5u/μl)	0.25-0.5 μl	1.25-2.5 u/20 μl reaction  (0.0625-0.125 u/μl)
Sterile ultrapure water	Up to 20 μl	N/A	Sterile ultrapure water	Up to 20 μl	N/A

 Similar setup is followed for Sybr Green based qPCR assays. Instead of Probe one may add Sybr Green in final concentrations of 0.2-2x. 

Recommended conditions: 

Step		Temperature			Time
Initial denaturation/Activation		95oC			15 min
3 steps program				Steps program
Denaturation	95oC		10-15 sec	Denaturation		95oC	10-15 sec
Annealing	45-68oC*		10-20 sec	Annealing/ extension		(Tm-1oC)*	30 sec
Extension	72oC		1min/kb  (6 sec for 100 bp ampliqons)

*Annealing temperature depends on primers’ Tm. For two steps programs the primers are designed to have a Tm ~ 68. For most primers sets tested, optimal Ta was found at 1-2^oC below the lower Tm of the primer set. 

Initial activation for 15 minutes is very important in the case of reaction with very low PCR template copies and for obtaining the maximum efficiency in qPCR setups (either TaqMan or SYBR Green chemistry).   DO NOT USE LOWER THAN 10 min!! 

2.One-Step RT qPCR 

PCR recommended reaction: 

One Step RT-qPCR with kit components			Taq man qPCR EQ protocol
Components	Standard 20μl assay	Final Concentration/  Quantity (20 μl assay)	Components	Standard 20μl assay	Final Concentration/  Quantity (20 μl assay)
10x Taq pol. Buffer w/o MgCl2	2μl	1x	5x One Step RT-qPCR buffer* (BR030)	5 μl	1x
25mM MgCl2	2 μl	1.5 -4 mM
dNTP mix 10mM each	0.4μl	0.2 mM each
10μΜ forward primer	0.8μl	0.2-0.8 μM  For single target 0.4 μM for multiplex 0.2 μΜ or lower	10μΜ forward primer	0.8 μl	0.2-0.8 μM  For single target 0.4 μM for multiplex 0.2 μΜ or lower
10μΜ reverse primer	0.8μl	0.2-0.8 μM  For single target 0.4 μM for multiplex 0.2 μΜ or lower	10μΜ reverse primer	0.8 μl	0.2-0.8 μM  For single target 0.4 μM for multiplex 0.2 μΜ or lower
10 μΜ Taqman Probe	0.4 μl	0.2-0.5 μM (~ half or lower than the primer conc.)	10 μΜ Taqman Probe	0.4 μl	0.2-0.5 μM (~ half or lower than the primer conc.)
Template DNA	Variable	10 fg-500 ng	Template DNA	variable	10 fg-500 ng
Hot Start Taq DNA pol. (5u/μl)	0.25-0.5 μl	1.25-2.5 u/20 μl reaction  (0.0625-0.125 u/μl)	Hot Start Taq DNA pol. (5u/μl)	0.25-0.5 μl	1.25-2.5 u/20 μl reaction  (0.0625-0.125 u/μl)
Engineered MMLV Reverse transcriptase, 200u/μl (RN001S)	0.05 μl	8-10 u/20 μL reaction  (0.4-0.5 u/μl)	Engineered MMLV Reverse transcriptase, 200u/μl  (RN001S)	0.05 μl	8-10 u/20 μL reaction  (0.4-0.5 u/μl)
Sterile ultrapure water	Up to 20 μl	N/A	Sterile ultrapure water	Up to 20 μl	N/A

Similar setup is followed for SYBR Green based qPCR assays. Instead of Probe one may add SYBR Green in final concentrations of 0.2-2x. 

One Step-RT recommended conditions: 

Step		Temperature			Time
Reverse Transcription step		55 oC			15 min
Initial denaturation/Activation of Taq DNA polymerase/Reverse Transcriptase deactivation		95oC			15 min
3 steps program (30-40 cycles)				2 Steps program (30-40 cycles)
Denaturation	95oC		10-15 sec	Denaturation		95oC	10-15 sec
Annealing	45-68oC*		10-20 sec	Annealing/ extension		(Tm-1oC)*	30 sec
Extension	72oC		1min/kb  (6 sec for 100 bp ampliqons)

If you are new user needing qPCR related user-friendly solutions:

1. Check a ready-to-use 2x SybrGreen qPCR mix with Hot Start properties (ΕQ 2x qPCR Μaster Μix Green kit, w/o ROX)
2. Check a ready-to-use, universal and very sensitive One Step RT kit