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End-Point PCR Kits

Platinum™ Green Hot Start PCR Master Mix (2X)

Invitrogen

Templates
/
/
Sample Amount
/
/
From
$ 73.75 (50 Reactions)
Sizes
3 (50 - 1000 Reactions)
Catalog IDs
13001-012, 13001-013, ...
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Real-Time PCR Kits

Score 2.50

Maxima Probe/ROX qPCR Master Mix (2X)

Thermo Scientific

Templates
Genomic DNA, Plasmid DNA, Viral DNA, cDNA
Genomic DNA, Plasmid DNA, Viral DNA, cDNA
From
$ 181.00 (200 Reactions)
Sizes
3 (200 - 4000 Reactions)
Catalog IDs
K0231, K0232, ...
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End-Point PCR Kits

2X One-Step RT-PCR Master Mix with Dual Dye

Norgen Biotek

Templates
Total RNA, mRNA
Total RNA, mRNA
Sample Amount
/
/
From
$ 243.00 (100 Reactions)
Sizes
3 (100 - 500 Reactions)
Catalog IDs
28242, 28273, ...
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Real-Time PCR Kits

Score 2.50

DyNamo ColorFlash Probe qPCR Kit

Thermo Scientific

Templates
Genomic DNA, cDNA
Genomic DNA, cDNA
From
$ 92.00 (100 Reactions)
Sizes
3 (100 - 2500 Reactions)
Catalog IDs
F-456S, F-456L, ...
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PCR Reagents

Score 6.20

Ribonuclease Inhibitor

Sigma-Aldrich

Reagent Type
Inhibitors
Inhibitors
Quantity
2500 Units, 10000 Units
2500 Units, 10000 Units
Applicable Processes
In vitro transcription, Real-time PCR, cDNA synthesis
In vitro transcription, Real-time PCR, cDNA synthesis
From
$ 136.00 (2500 Units)
Sizes
2 (2500 - 10000 Units)
Catalog IDs
R1158-2.5KU, R1158-10KU
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Competent Cells

Score 10.00

Stellar™ Competent Cells

Clontech

Applicable Processes
Molecular cloning
Molecular cloning
From
$ 185.00 (1000 µl)
Sizes
3 (1000 - 5000 µl)
Catalog IDs
636763, 636767, ...
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Real-Time PCR Kits

Score 10.00

SYBR GreenER™ qPCR SuperMix Universal

Invitrogen

Templates
Genomic DNA, Plasmid DNA, cDNA
Genomic DNA, Plasmid DNA, cDNA
From
$ 231.00 (100 Reactions)
Sizes
3 (100 - 2000 Reactions)
Catalog IDs
11762-100, 11762-500, ...
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End-Point PCR Kits

Score 7.20

AmpliTaq® 360 DNA Polymerase

Applied Biosystems

Templates
Genomic DNA
Genomic DNA
Sample Amount
/
/
From
$ 64.00 (1 Unit)
Sizes
7 (1 - 25000 Unit)
Catalog IDs
4398808, 4398818, ...
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Real-Time PCR Kits

Score 3.30

Maxima SYBR Green/Fluorescein qPCR Master Mix (2X)

Thermo Scientific

Templates
Genomic DNA, Plasmid DNA, Viral DNA, cDNA
Genomic DNA, Plasmid DNA, Viral DNA, cDNA
From
$ 173.00 (200 Reactions)
Sizes
3 (200 - 4000 Reactions)
Catalog IDs
K0241, K0242, ...
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Molecular Cloning

Score 0.80

DNA Ligation Kit, Version 2.1

Clontech

Cloning Type
Ligation
Ligation
Insert Type
DNA
DNA
Cloning Time
> 3 min
> 3 min
From
$ 187.00 (50 Reactions)
Sizes
1 (50 Reactions)
Catalog IDs
6022
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Real-Time PCR Kits

Score 5.60

Luminaris HiGreen qPCR Master Mix

Thermo Scientific

Templates
Genomic DNA, Plasmid DNA, Viral DNA, cDNA
Genomic DNA, Plasmid DNA, Viral DNA, cDNA
From
$ 181.00 (250 Reactions)
Sizes
4 (250 - 5000 Reactions)
Catalog IDs
K0991, K0992, ...
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End-Point PCR Kits

Score 10.00

Platinum® PCR SuperMix High Fidelity

Invitrogen

Templates
Genomic DNA
Genomic DNA
Sample Amount
/
/
From
$ 266.00 (100 Reactions)
Sizes
2 (100 - 5000 Reactions)
Catalog IDs
12532-016, 12532-024
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After sample preparation, your target molecule will most likely require analysis. If your sample volume is too small, then amplification (via PCR or molecular cloning) is necessary to allow accurate downstream evaluation. Additionally, separation by electrophoresis can your identify specific species or fragments within your sample.

Problems in the lab? Find the solutions in our Knowledge and Troubleshooting sections. Alternatively, our Community of experienced researchers can help! Still confused? See How ZAGENO Works.

Amplification - Polymerase Chain Reaction (PCR)

One of the classic laboratory techniques in all of biology, ranging from ecology and evolution to biotechnology, is the polymerase chain reaction. PCR is an in vitro method for the amplification of a specific DNA template to quickly produce a large number of copies. Initially developed in 1983 by Kary Mullis; PCR helped replace the slower cloning techniques which required days of lab work.

PCR Reaction Components:

1.     DNA or RNA template: which we want to amplify.

2.     Nucleotides (dCTP, dGTP, dATP, dGTP): DNA and RNA building blocks, required for elongation

3.     Primers: short nucleotide strands which anneal to the positions that we want to start and end the amplification.

4.     DNA polymerase: the enzyme catalyzing the reaction.

5.     Accessory additives like buffers and Mg2+

Amplification Steps:

1.     Denaturation: DNA is heated in a thermocycler to split the double-stranded molecule into two single strands.

2.     Hybridization: Primers can anneal to their target sequences on the single-stranded DNA, during a cool-down phase.

3.     Elongation: The single strands of DNA are extended via polymerases, which start from the primer sequences, resulting in two double-stranded DNA molecules.

4.     The above three steps repeat until there is a lack of substrate or the product accumulation halts the reaction. After every cycle, the amount of product increases exponentially.

PCR was initially established to detect particular DNA  sequences within a sample, a technique still commonly used today. However, many other applications have been elaborated over the past few decades. Nowadays different types of PCR exist:

· End-Point

· Real Time (qPCR) (quantitative)

· Reverse Transcription (RT-PCR)

Other variations include multiplex, digital and nested PCR. Additionally, adapted polymerases used in PCR may incorporate features such as a hot start mechanism or proofreading activity. Not only is PCR a key method for DNA replication, but it is also often used in cloning processes.

Amplification - Molecular Cloning

Despite the name, cloning is the process of copying either segments or the entire genome by utilizing the cell’s natural self-replication steps; simply, cloning refers to the procedure of making multiple copies of recombinant DNA. These molecular cloning methods allow for scientists to build a bank of DNA to work from, which are commonly referred to as DNA libraries.

In sum, molecular cloning steps typically involve the choice of a host cell and cloning vector, preparation of vector and target DNA, formation of recombinant DNA, transfection of recombinant DNA into a host cell, then selection and screening of host cells for target DNA.

Molecular cloning is similar to PCR, but rather than replicate DNA in vitro; cloning occurs within a living cell. However, molecular cloning can sometimes utilize PCR for preparing DNA that requires cloning.

Separation - Electrophoresis

The final stages of PCR and some methods of nucleic acid purification require electrophoresis; a common technique employed in laboratories to filter molecules based on size. In principle, electrophoresis separates particles based on electrical charge.

For example, DNA molecules are negatively charged. So, to separate them based on size, a positive electrical current is passed through a gel; forcing the DNA molecules to travel through the gel at a velocity inversely proportional to their size, depending on the gel porosity. So larger molecules will have a more difficult time to pass through the gel when compared to smaller molecules, thus allowing for separation between DNA molecules.