Hot Start Taq DNA polymerase is a thermostable DNA polymerase manufactured with high sensitivity, specificity, and DNA-free traits. It is certified as RNase, DNase, and E. coli DNA free, that guarantees 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. 

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:

 PCR Guidelines:

 Reactions may be set at room temperature.

PCR recommended reaction:

* 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:

*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 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 post production

Highly robust detection of targets from human genomic DNA, even with minute amounts of enzyme units per reaction vs a competitor that uses the same chemistry for Hot start Taq DNA pol. preparation.

Other applications:

1. qPCR on DNA templates

PCR recommended reaction: 

*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: 

*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 8^oC below the Tm temp. Do not incubate lower than 55 ^oC, to avoid lower polymerase activity!

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: 

*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: 

*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 8^oC below the Tm temp. Do not incubate lower than 55 ^oC  to avoid lower polymerase activity!

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

1. Check ready-to-use 2x  SybrGreen qPCR  mix with Hot Start properties check RN014S kit (ΕQ 2x qPCR Μaster Μix Green kit, w/o ROX (TM))
2. Check the One-step RT qPCR analysis kit (RN010S) the highly sensitive kit for RNA analysis in one single reaction.
3. ((((Check out/ Stay tuned for our universal qPCR kit for more flexibility in your DNA/RNA quantification studies (this product may be released in parallel with Sheep/Goat Pox kit or as a separate entity))))