HotStart Taq DNA Polymerase basic kit, 500 U

HotStart Taq DNA Polymerase basic kit, 500 U

Hot Start Taq DNA polymerase is a thermostable DNA polymerase manufactured with the highest standards. It is certified as RNase, DNase and E.coli DNA free, that guarantee reproducible and efficient PCR reactions.

Taq DNA Polymerase is a thermostable enzyme that catalyzes 5’→3′ synthesis of DNA. The enzyme has no detectable 3’→5′ proofreading exonuclease activity but possesses low 5’→3′ exonuclease activity. Hot Start Taq DNA Polymerase has high processivity up to 5 kb, while it can reliably amplify a substrate as low as 6-12 copies per reaction.

(SKUs: PD012S)

100.00

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 72oC.

Reagents supplied:

PD012S kit
Cat NoDescriptionTubes No.
PD002-0.100HotStart 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
BR006-1.410x Taq DNA polymerase Buffer with 15mM MgCl2 (100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15mM MgCl2)1
BR007-1.410x Taq DNA polymerase Buffer w/o MgCl2 (100 mM Tris-HCl (pH 8.3), 500 mM KCl)1
BR008-1.425mM MgCl21
BR028-250dNTPs mix 10mM each, 0.250 ml1
BR033-1.8Nuclease Free Water, 1.8 ml2

PCR Guidelines:

Reactions may be set at room temperature.

PCR recommended reaction:

Components25μl assayFinal Concentration/ Quantity (25μl assay)50μl assayFinal Concentration/ Quantity (50μl assay)
10x Taq pol. Buffer with 15mM MgCl22.5μl1x5μl1x
dNTP mix 10mM each0.5μl0.2 mM each1μl0.2 mM each
10μΜ forward primer0.5μl0.2-0.8 μM1 μl0.2-0.5 μM
10μΜ reverse primer0.5μl0.2-0.8 μM1 μl0.2-0.5 μM
Template DNAVariable10 fg-500 ng*Variable100 fg-500 ng*
Hot Start Taq DNA pol. (5u/μl)0.25μl1.25 u/25 μl reaction (0.05 u/μl)0.25-0.5μl1.25-2.5 u/50 μl reaction (0.025-0.05 u/μl)
Sterile ultrapure waterUp to 25 μl Up to 50 μl 
* Substrate quantity is mostly dependent on the complexity and the purity of substrate and should be defined after testing for each primer set/substrate combination. For highly pure DNA substrates (OD260/OD280 ~ 2 and OD260/OD230~ 2,2) dissolved in water for molecular biology, 5-10 copies of specific target substrate are enough for end product gel visualization.

PCR recommended conditions:

StepTemperatureTime
Initial denaturation/Activation95oC15 min
25-40 cycles
Denaturation95oC30sec
Annealing45-68oC*20sec
Extension72oC1min/kb
   
Final extension72oC5 min
Hold4-12oCIndefinitely
*Annealing temperature depends on primers’ Tm

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 -20οC ± 5οC

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 
*BR030 may be separately purchased. It contains an EQ optimized chemistry for qPCR applications along with dNTPs. 

 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 95o10-15 sec Denaturation 95o10-15 sec 
Annealing 45-68oC* 10-20 sec Annealing/ extension (Tm-1oC)* 30 sec 
Extension 72o1min/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 1-2oC below the lower Tm of the primer set. 

*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 1-2oC 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 
*BR030 may be separately purchased. It contains an EQ optimized chemistry for qPCR applications along with dNTPs.  RN001S is part of the RN012S kit, which may be separately purchased

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 o15 min 
Initial denaturation/Activation of Taq DNA polymerase/Reverse Transcriptase deactivation95oC 15 min 
3 steps program (30-40 cycles) 2 Steps program (30-40 cycles) 
Denaturation 95o10-15 sec Denaturation 95o10-15 sec 
Annealing 45-68oC* 10-20 sec Annealing/ extension (Tm-1oC)* 30 sec 
Extension 72o1min/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 1-2oC below the lower Tm of the primer set. 

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 

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