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Biotechrabbit

[Botechrabbit] 2X Hot-Start PCR Master Mix

Cat-No. BR0200205

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제품 설명

2X Hot-Start PCR Master Mix

제품 번호

BR0200205 - 200 reactions of 50 µl

제품 특징

Description

biotechrabbit™ UPstart Plus Taq Antibody is an ultra-pure monoclonal antibody against the Taq DNA polymerase. It is produced in cell culture to ensure highest quality.

The antibody can be used with highly efficient Taq DNA polymerases, provides an excellent method for “hot start” PCR and enhances PCR specificity and sensitivity. PCR hot start prevents the formation of primer–dimers and nonspecific amplification and allows convenient PCR setup at room temperature.

In the first denaturation step of the thermal cycling, the UPstart Pus Taq Antibody becomes nonfunctional and the active Taq DNA polymerase is released. This antibody-mediated hot-start method is significantly more convenient to use than other hot-start methods. Polymerase reactivation using this antibody is faster than with methods using chemically inhibited polymerases.

Other Information

Component	Composition
UPstart Taq Antibody Plus	UPstart Taq Antibody Plus, 1 mg/ml (5 U/µl) in storage buffer containing 50% (v/v) glycerol

STORAGE

−20°C (until expiry date – see product label)

Unit Definition

One unit is defined as the amount of antibody required to inhibit >95% of 1 unit Taq DNA polymerase at 60°C. One unit UPstart Plus Taq Antibody (200 ng of the antibody) will ensure inhibition of Taq DNA polymerase.

Quality Control

Functional Assay

UPstart Plus Taq Antibody was functionally tested on hot-start PCR.

Inhibition activity

The Taq DNA polymerase activity was inhibited to >95% at 60°C when adding 200 ng UPstart Plus Taq Antibody (1 U) to one unit Taq DNA polymerase.

Mouse genomic DNA contamination assay

An UPstart Plus Taq Antibody sample is analyzed for the presence of contaminating mouse genomic DNA. No mouse genomic DNA was detectable.

Protocols

Prevention of PCR contamination

When assembling the amplification reactions, care should be taken to eliminate the possibility of contamination with undesired DNA.

Use separate clean areas for preparation of samples and reaction mixtures and for cycling.
Wear fresh gloves. Use sterile tubes and pipette tips with aerosol filters for PCR setup.
Use only water and reagents that are free of DNA and nucleases.
With every PCR setup, perform a contamination control reaction that does not include template DNA.

Pre-mix preparation of Taq DNA polymerase and UPstart Plus Taq Antibody

A pre-mix will result in a hot-start Taq DNA polymerase that can be used for the setup of hot-start PCR reactions. An example for a pre-mix is given below.

Component	Volume	FINAL CONCENTRATION
Taq DNA Polymerase, 5 U/µl (i.e. BR0100101)	20 µl	100 U
UPstart Plus Taq Antibody	20 µl	100 U
Gives 100 reactions with 1 unit Hot-start Taq DNA polymerase per reaction. For one reaction use:
Taq DNA Polymerase / UPstart Plus Taq Antibody pre-mix	0.4 µl	1 U Hot-Start Taq DNA Polymerase

Mix and centrifuge briefly to collect the liquid in the bottom of the tube.
The mix can be stored at -20°C for up to 6 months.

Basic Protocol

Thaw on ice and mix all reagents well, especially the MgCl₂ solution and dNTPs.
Keep all reagents and reactions on ice.
When setting up multiple reactions, prepare a master mix of water, buffer, dNTPs, UPstart Plus Taq antibody and Taq DNA polymerase. Prepare enough master mix for one more than the actual number reactions. Alternatively, use biotechrabbit Hot-Start PCR Master Mix, 2× (cat. no. BR0200201)
Pipet the master mix into thin-walled 0.2 ml PCR tubes.
Add template and primers separately if they are not used in all reactions.

Component	Volume	Final concentration
Taq DNA Polymerase, 5 U/µl	0.2 µl	1 U
UPstart Plus Taq Antibody	0.2 µl	1 U
Mix Taq DNA polymerase and UPstart Taq Antibody and incubate at room temperature for 15 min. Alternatively use pre-mixed Taq DNA polymerase and UPstart Taq Antibody.
10× Reaction Buffer	5 µl	1×
50 mM MgCl₂	Variable (standard 1.5 µl)	1.5 mM
	Higher than 2 mM MgCl₂ might increase yield but reduce fidelity
5× PCR Enhancer (optional)	10 µl	1×
10 mM dNTP Mix	1 µl	200 µM
Forward primer	Variable	0.2–1 µM
Reverse primer	Variable	0.2–1 µM
Template DNA	Variable	10 pg–1 μg
	Use 0.01–1 ng for plasmid or phage DNA and 0.1–1 μg for genomic DNA
Nuclease free water	Variable
Total volume	50 µl

Mix and centrifuge briefly to collect the liquid in the bottom of the tube.
Place in the PCR cycler.

Cycling Program

Step	Temperature	Time	Cycles
Initial activation	94°C	3–5 min	1
Denaturation	94°C	30 s	25–35
Annealing	55°C	15–30 s	25–35
	Approximately 5°C below T_m of primers
Extension	72°C	30–60 s/kb	25–35
Final extension	72°C	5 min	1
	To extend all incomplete PCR products
Storage in the cycler	4°C	Indefinitely	1

Add loading dye solution (see DNA Loading Dye, 6×, cat. no. BR0800301) to the reactions to analyze PCR products on a gel or store them at −20°C.

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