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Amplification & Separation on ZAGENO

FastStart Universal SYBR Green Master (Rox) Roche Life Science
Templates Genomic DNA, Plasmid DNA, cDNA
From $ 321.00 (500 Reactions)
Sizes 2 (500 - 5000 Reactions)
Catalog IDs 04913850001, 04913914001
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pTWV228 DNA (0.5 OD) Clontech
Quantity 25 µg Applicable Processes Molecular cloning, Sequencing
From $ 61.00 (25 µg)
Sizes 1 (25 µg)
Catalog IDs 3333
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2X One-Step RT-PCR Master Mix with Dual Dye Norgen Biotek
Templates Total RNA, mRNA Sample Amount /
From $ 243.00 (100 Reactions)
Sizes 3 (100 - 500 Reactions)
Catalog IDs 28242, 28273, ...
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Ribonuclease Inhibitor Sigma-Aldrich
Reagent Type Inhibitors Quantity 10000 Units, 2500 Units Applicable Processes 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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Stellar™ Competent Cells Clontech
Applicable Processes Molecular cloning
From $ 185.00 (1000 µl)
Sizes 3 (1000 - 5000 µl)
Catalog IDs 636763, 636767, ...
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SYBR GreenER™ qPCR SuperMix Universal Invitrogen
Templates 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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AmpliTaq® 360 DNA Polymerase Applied Biosystems
Templates Genomic DNA Sample Amount /
From $ 64.00 (1 Unit)
Sizes 7 (1 - 25000 Unit)
Catalog IDs 4398808, 4398818, ...
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DNA Ligation Kit, Version 2.1 Clontech
Cloning Type Ligation Insert Type DNA Cloning Time > 3 min
From $ 187.00 (50 Reactions)
Sizes 1 (50 Reactions)
Catalog IDs 6022
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Platinum® PCR SuperMix High Fidelity Invitrogen
Templates Genomic DNA Sample Amount /
From $ 266.00 (100 Reactions)
Sizes 2 (100 - 5000 Reactions)
Catalog IDs 12532-016, 12532-024
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REDExtract-N-Amp™ Blood PCR Kit Sigma-Aldrich
Templates Genomic DNA Sample Amount /
From $ 33.50 (10 Reactions)
Sizes 4 (10 - 1000 Reactions)
Catalog IDs XNABS-1KT, XNAB-1KT, ...
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Gateway® pENTR™ 1A Dual Selection Vector Invitrogen
Cloning Type Gateway Insert Type Attachment sites flanked DNA Cloning Time /
From $ 209.00 (10 µg)
Sizes 1 (10 µg)
Catalog IDs A10462
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EXTENDER™ PCR-TO-GEL MASTER MIX, 2X AMRESCO
Templates Genomic DNA Sample Amount /
From $ 49.00 (50 Reactions)
Sizes 2 (50 - 100 Reactions)
Catalog IDs N867-1.25ML, N867-2X1.25ML
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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.