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GC-Rich PCR Performance

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INVSc1, S. cerevisiae Yeast Strain Invitrogen™

The S. cerevisiae strain, INVSc1, is a fast-growing diploid strain ideal for expression. INVSc1 has the following genotype:

INVSc1: MATa his3D1 leu2 trp1-289 ura3-52 MAT his3D1 leu2 trp1-289 ura3-52

ViraPower™ HiPerform™ Promoterless Gateway™ Vector Kit Invitrogen™

The ViraPower™ HiPerform™ Promoterless Gateway® Vector Kit contains the MultiSite Gateway®-adapted ViraPower™ HiPerform™ promoterless lentiviral expression vector, pLenti6.4⁄R4R2⁄V5-DEST™ for easy recombination-based cloning and lentiviral-based high-level expression of a target gene from any promoter of choice in dividing and non-dividing mammalian cells. The pLenti6.4⁄R4R2⁄V5-DEST™ vector is equipped with two key genetic elements, making it a HiPerform™ vector: the Woodchuck Posttranscriptional Regulatory Element (WPRE) and the central Polypurine Tract (cPPT) sequence from the HIV-1 integrase gene to produce at least 4-fold increase in protein expression compared to vectors lacking these elements.

• Generates replication-incompetent lentivirus for transducing dividing and non-dividing mammalian cells
• Easy, simultaneous, recombination-based cloning of multiple DNA fragments in a defined order and orientation using MultiSite Gateway® technology
• Expression of the target gene under the control of a promoter of choice
• Stable, long-term expression
• Enhanced protein expression, up to 4-fold or greater, compared to traditional lentiviral expression systems

Key Features
• WPRE from the woodchuck hepatitis virus, increases transgene expression and cPPT from the HIV-1 integrase gene, increases the copy number of lentivirus integrating into the host genome, thus increasing viral titer. WPRE and cPPT together produce at least a four-fold increase in protein expression in most cell types, compared to other vectors that do not contain these elements.
• Promoterless vector to express the target gene under the control of a promoter of choice
• Blasticidin selection marker for stable selection under control of PGK promoter for long-term, persistent expression

Kit includes
• pLenti6.4⁄R4R2⁄V5-DEST™ Kit
• pENTR™ 5’ TOPO® TA Cloning Kit (Cat # K59120)
• One Shot® Stbl3™ Chemically Competent E. coli (Cat # C737303)
• pENTR® Gus positive control plasmid

For research use only. Not intended for any therapeutic or diagnostic use.

iPrep™ Card: gDNA Tissue Invitrogen™

The iPrep™ Protocol Card is a flash, 512 KB memory card. Each iPrep™ Protocol card is pre-programmed with the purification protocol that directs the volume of reagents used and incubation time.
The following iPrep™ Protocol Cards are available from Invitrogen:
- iPrep™ Forensic Card for use with the iPrep™ ChargeSwitch® Forensic Kit and the iPRep™ ChageSwitch® gDNA Buccal kit
-iPrep™ Tissue Card for use with the iPrep ChargeSwitch® Tissue Kit
- iPrep™ Blood Card for use with the iPrep PureLink Blood kit

PureLink™ RNA Mini Kit Invitrogen™

The PureLink RNA Mini Kit is a column-based kit used to isolate high-quality total RNA from a wide variety of sample types in 20 minutes using standard laboratory equipment. The kit includes RNase-free lysis and wash solutions that protect RNA from RNases while liberating the RNA from DNA, proteins, and other cellular debris. The fast spin-column workflow is ideal for processing low to mid-throughput batch sizes. The advanced PureLink RNA Mini spin column design allows for maximum sample input (200 mg of tissue) and RNA recovery (up to 1000 µg). This means that users can process both small and large sample sizes with the same RNA isolation kit.

• Process sample inputs from 5–200 mg of animal/plant tissue or from 0.5 x 106 to 108 tissue culture cells with one spin column in 20 minutes
• Isolate more RNA than competitor mini spin columns: up to 1000 µg of total RNA from one column
• Use isolated RNA in most applications without the need for further DNase treatment (very low minimal residual DNA carry over)
• Flexible–optional on-column or post-isolation DNase treatment is available with PureLink DNase Set for easy removal of residual DNA

Extra-large binding capacity enables rapid RNA purification using standard laboratory equipment
The PureLink RNA Mini Kit provides rapid purification of total RNA from a wide range of cells and tissue types to yield up to 1000 µg of purified RNA from a single extraction (see Figure 1). High-quality total RNA can be obtained from mini- to midi-prep amounts of starting material with just trace amounts of residual genomic DNA contamination. The extra-large binding capacity enables one kit to handle most RNA isolation needs in a quick 20 minute protocol without the need for special sample processing instruments.

Easy, optional, on-column DNase treatment for sensitive applications
The PureLink RNA Mini Kit columns are highly efficient for isolating high quality total RNA while removing the majority of genomic DNA. In general, most applications requiring RNA from animal tissue or mammalian cell lines do not require additional DNase treatment. However, some applications such as gene expression analysis by qRT-PCR without intron-spanning primers or working with samples from organisms with very small or no introns may require more complete removal of residual contaminating DNA. The PureLink DNase Set allows for convenient on-column digestion of DNA during the RNA isolation protocol. Treating with DNase while “on-column” is easier and allows higher RNA recovery than treating with DNase after the RNA has been isolated. The PureLink DNase Set can also be used to remove residual DNA from RNA that has been previously purified. Both the “on-column” and post-RNA purification workflows are options available with the PureLink DNase Set.

Simplified, non-toxic RNA purification
The PureLink RNA Mini Kit utilizes non-toxic guanidine-isothiocyanate lysis buffer to protect the RNA during the isolation steps. Special RNase-free reagents combined with certified RNase-free silica membranes in a unique column configuration allow for a safe and easy procedure that can typically be completed in less than 20 minutes without the need of hazardous phenol/chloroform extraction, CsCl centrifugation, or LiCl or alcohol precipitation.

The PureLink RNA Mini Kit is recommended for use with the Homogenizer (Cat. No. 12183026), designed to homogenize cell or tissue lysates via centrifugation, prior to nucleic acid purification. The Homogenizer is especially effective for clarifying particulates from plant tissues.

For plant tissues rich in polyphenolics or starch (e.g., pine needles, potato tubers), we recommend using PureLink Plant RNA Reagent (Cat. No. 12322012) together with the PureLink RNA Mini Kit.

Platinum™ II Taq Hot-Start DNA Polymerase Invitrogen™

Invitrogen Platinum II Taq Hot-Start DNA Polymerase is designed for universal primer annealing and fast, easy PCR with its unique combination of innovative buffer, high-performance engineered Taq DNA polymerase, and superior hot-start technology.

Features of Platinum II Taq Hot-Start DNA Polymerase include:
Innovative buffer—enables universal annealing temperature by isostabilizing primer-template duplex structures
Engineered Taq DNA polymerase—confers fast cycling and resistance to common inhibitors
Platinum hot-start technology—enables superior specificity, sensitivity, and yields; allows for room temperature reaction setup

Platinum II Taq Hot-Start DNA Polymerase is an engineered Taq DNA polymerase that shows increased resistance to reaction inhibitors originating from sample material or DNA purification steps. The polymerase has a higher DNA synthesis rate and delivers PCR results more than two times faster than other Taq DNA polymerases. Proprietary Platinum Taq antibodies block polymerase activity at ambient temperatures and dissociate after the initial denaturation step at 94°C. This automatic 'hot start' provides increased sensitivity, specificity, and yield, while allowing reaction assembly at room temperature.

Due to the unique composition of the Platinum II PCR buffer, the annealing temperature is 60°C for most primer pairs designed following the general design rules. Isostabilizing molecules in the buffer increase primer–template duplex stability during the annealing step and contribute to enhanced specificity without the need to optimize annealing temperature for each primer pair. With Platinum II Taq Hot-Start DNA Polymerase, different PCR assays can be cycled together using the same protocol with universal primer annealing temperature and the extension step selected for the longest fragment to be amplified.

Platinum II Taq Hot-Start DNA Polymerase is provided with the optional Platinum GC Enhancer for specific amplification and improved yields of GC-rich targets.

Use Platinum II Taq Hot-Start DNA Polymerase for the amplification of DNA from complex genomic, viral, and plasmid templates, as well as in RT-PCR, in applications like genotyping, high-throughput PCR, or with samples of suboptimal purity.

For increased convenience, we offer Platinum II Hot-Start PCR Master Mix (2X), where Platinum II Taq Hot-Start DNA Polymerase is provided in a ready-to-use mixture with Platinum II PCR buffer and dNTPs, thus reducing the number of pipetting steps during PCR reaction setup. Platinum II Hot-Start Green PCR Master Mix (2X) is also available, which additionally contains a density reagent and two tracking dyes for direct loading of PCR products on gels, further streamlining the PCR workflow from setup to final analysis of the result.

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Total RNA Lysis Solution, Nucleic Acid Purification Invitrogen™

Total RNA Lysis Solution is a 2X concentrate ideal for the chemical disruption of cells and tissue (cultured cells, primary cell isolates, and animal/plant tissues) prior to RNA or DNA extraction. Following downstream purification (e.g. using the 6100 PrepStation), intact RNA can be isolated that is free of contaminating protein and metal ions. Total RNA Lysis solution is the recommended reagent for the Total RNA Isolation Chemistry System for the ABI PRISM™ 6100 Nucleic Acid PrepStation.

For research use only. Not for use in diagnostic procedures.

DNAzol™ Reagent, for isolation of genomic DNA from solid and liquid samples Invitrogen™

DNAzol® Reagent is a complete and ready-to-use organic reagent for the isolation of genomic DNA from solid and liquid samples of animal, plant, yeast, and bacterial origin. The DNAzol® Reagent procedure can be completed in typically 10–30 minutes with DNA recoveries of 70–100%. DNAzol® Reagent is:

Versatile—can use with a broad spectrum of sample materials
Efficient—rapid isolation and high recovery of genomic DNA

Efficient isolation of genomic DNA from a variety of sample types
The DNAzol® Reagent procedure uses a novel guanidine-detergent lysing solution that permits selective precipitation of DNA from cell lysate. 1 mL of DNAzol® Reagent can be used to isolate genomic DNA from 1–3 x 107 cells, from 0.1 mL of whole blood, or from 25–50 mg tissue.

Rapid isolation and high recovery of genomic DNA
The DNAzol® Reagent combines reliability, efficiency, and simplicity in its DNA isolation protocol. During isolation, a biological sample is lysed (or homogenized) in DNAzol® Reagent, and the genomic DNA is then precipitated from the lysate with ethanol. Following an ethanol wash, DNA may be solubilized in either water or 8 mM NaOH. The entire procedure can be completed in 10–30 min with DNA recovery of 70–100%.

Isolated DNA can be used for a number of downstream applications
Genomic DNA extracted using the DNAzol® procedure is suitable for a variety of applications, including Southern blotting, cloning, PCR, restriction endonuclease digestion, and dot blot hybridization.

iPrep™ Card: Total RNA Invitrogen™

The iPrep™ Protocol Card is a flash, 512 KB memory card. Each iPrep™ Protocol card is pre-programmed with the purification protocol that directs the volume of reagents used and incubation time.

Yeast Nitrogen Base Invitrogen™

Yeast nitrogen base (YNB) is a base medium for preparation of minimal and synthetic defined yeast media. YNB contains ammonium sulfate but does not contain amino acids. Both bulk and convenient single-use pouches are available.

MPC™-6 (Magnetic Particle Concentrator) Thermo Scientific™

The MPC™-6 (Magnetic Particle Concentrator) holds six tubes with variable diameters (10–30 mm). Optimal working volume: 5–15 mL. Magnetic particle concentrators are designed to obtain optimal binding of Dynabeads™ with targets (e.g., cells, proteins, and nucleic acids).

KingFisher™ Cell and Tissue DNA Kit Thermo Scientific™

Rapidly purify high-quality, reproducible DNA from cell and tissue samples, as well as cultured bacteria, with the Thermo Scientific™ KingFisher™ Cell and Tissue DNA Kit. This unique nucleic acid purification workflow provides an optimized, high-throughput method for extreme flexibility.

PCR Master Mix (2X) Thermo Scientific™

Thermo Scientific PCR Master Mix is a 2X concentrated solution of Taq DNA Polymerase, dNTPs, and all of the components required for PCR, except DNA template and primers. This pre-mixed formulation saves time and reduces contamination due to a reduced number of pipetting steps required for PCR set up. The mix is optimized for efficient and reproducible PCR.


Convenient, ready-to-use mix
Thermostable—half life is more than 40 min at 95°C
Generates PCR products with 3'-dA overhangs
Incorporates modified nucleotides (e.g., biotin-, digoxigenin-, fluorescently-labeled nucleotides)


• Routine PCR amplification of DNA fragments up to 5 kb
• High throughput PCR
• DNA labeling


• The error rate of Taq DNA Polymerase in PCR is 2.2 x 10-5 errors per nt per cycle, as determined by a modified method described in. Accordingly, the accuracy of PCR is 4.5 x 104. Accuracy is an inverse of the error rate and shows an average number of correct nucleotides incorporated before an error occurs.

ChargeSwitch™ Plasmid Yeast Mini Kit Invitrogen™

Sample size 1 ml S. cerevisiae culture; Typical yield up to 25 ng

• Increased transformation efficiency
• Reagents that avoid PCR inhibition
• Purifies DNA with improved downstream performance

The ChargeSwitch® Plasmid Yeast Mini Kit provides an efficient method for purifying high-quality plasmid DNA from 1 ml of Saccharomyces cerevisiae grown in selective minimal media. This kit typically yields up to 25 ng of plasmid DNA. Data generated from transformation and PCR experiments using ChargeSwitch® purified DNA shows improved performance versus DNA purified by other methods (Figure 1). This kit includes all the necessary reagents for the number of preps listed and requires the use of a magnetic accessory. Please see the Accessories Chapter (Chapter 6) to select the best magnetic accessory for your needs.

EKMax™ Enterokinase Invitrogen™

EKMax™ is a recombinant preparation of the catalytic subunit of bovine enterokinase (1). EKMax™ recognizes the sequence Asp-Asp-Asp-Asp-Lys and cleaves the peptide bond after the lysine residue. The enzyme can be used to cleave any fusion protein that carries this peptide sequence (Figure 1).

Application: Removal of fusion tags from recombinant proteins.

Unit Definition: One unit of EKMax™ is the amount of enzyme required to digest 20 µg of a thioredoxin-CAT fusion protein to 90% completion in 16 hours at 37°C. One EKMax™ unit is equivalent to ~190 trypsinogen activation units.

pcDNA™3.1/Hygro(-) Mammalian Expression Vector Invitrogen™

This pcDNA™3.1/Hygro(-) vector is designed for high-level, constitutive expression in a variety of mammalian cell lines. It contains a Hygromycin selectable marker and a reverse-orientation multiple cloning site.

The pcDNA™3.1 Expression Vector Family
Three untagged versions of pcDNA™3.1 (available separately), each with a different selectable marker (Geneticin®, Zeocin™, or Hygromycin), are for use alone or in co-transfections. All three vectors offer the following features:
• Cytomegalovirus (CMV) enhancer-promoter for high-level expression
• Large multiple cloning site in either forward (+) or reverse (-) orientations
• Bovine Growth Hormone (BGH) polyadenylation signal and transcription termination sequence for enhanced mRNA stability
• SV40 origin for episomal replication and simple vector rescue in cell lines expressing the large T antigen (i.e., COS-1 and COS-7)
• Ampicillin resistance gene and pUC origin for selection and maintenance in E. coli
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