How long is the Ion Chef™ run for the Ion 540™ Kit - Chef?

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Two Ion 540™ chips can be prepped and loaded in 13 hours, with only 15 minutes of hands on time.

Answer Id: E13147

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What version of Torrent Suite™ Software is compatible with the Ion 540™ Kit - Chef?

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The Ion 540™ Kit - Chef is compatible with Torrent Suite™ Software v5.0 and above.

Answer Id: E13148

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At what pressure should I set the gas regulator for my Ion PGM™ Sequencer? When should I change out my tank?

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Please ensure that the gas pressure going to your instrument is set to 30 PSI. Monitor the pressure in your tank closely, and exchange with a fresh tank when the pressure goes down to 500 PSI.

Answer Id: E6874

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The original Invitrogen™ Qubit™ (1.0) Fluorometer could be connected to a computer. Is that also true of the Invitrogen™ Qubit™3.0 Fluorometer?

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Yes, the Qubit 3.0 Fluorometer comes with both a USB and cable to connect to a computer.

Answer Id: E10254

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For starting my first Protein Thermal Shift™ experiment, what experimental ranges should I use for dye/protein concentration and such?

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Answer

For initial experiments, we suggest the following ranges as a starting point:

Protein Thermal Shift™ Dye: Use the dye at 1X-20X
Total volume per reaction: 10-50 μL
Total protein per reaction: ~0.05-5 μg (average 1 μg)
Typical thermal profile: 25°C-99°C

Set up the reaction on ice and start the run as soon as convenient. The Protein Thermal Shift™ Dye stability at 4°C and room temperature is at least 24 hours, in the dark.

Answer Id: E7577

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What is the codon usage for Pichia?

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It is doubtful codon usage plays as great a role in general as is commonly believed. Translation initiation is probably more of a rate limiting step than elongation.

Use the following codon usage list to design your gene in the order of preference:

Glycine: GGT or GGA
Glutamic acid: GAG or GAA
Aspartic acid: GAC or GAT
Valine: GTT or GTC
Alanine: GCT or GCC
Arginine: AGA or CGT
Serine: TCT or TCC
Lysine: AAG
Aspargine: AAC
Methionine: ATG
Isoleucine: ATT or ATC
Threonine: ACT or ACC
Tryptophan: TGG
Cysteine: TGT
Tyrosine: TAC
Leucine: TTG or CTG
Phenylalanine: TTC
Glutamine: CAA or CAG
Histidine: CAC or CAT
Proline: CCA or CCT

Answer Id: E3746

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Can I use the Ion 540™ Kit - Chef to prepare samples from the Ion 16S™ Metagenomics Kit?

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No, the Ion 16S™ Metagenomics Kit utilizes 400 bp reads, so it is incompatible with the Ion 540™ Kit - Chef, which only yields 200 bp reads.

Answer Id: E13149

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What is the thermal output of the Ion PGM™ System?

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The Ion PGM™ System typically draws ~200 to 300 W and outputs ~682 to 1023 BTU/h.

Answer Id: E6875

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Can I use my old Invitrogen™ Quant-iT™ Kits labeled “for use with Invitrogen™ Qubit™ Fluorometer” with the Invitrogen™ Qubit™ 2.0 and 3.0 Fluorometers?

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Answer

Yes, these kits will work with all Qubit Fluorometers.

Answer Id: E10255

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What aspects of cell culture media can be customized?

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The Gibco™ Media Configurator allows you to add, remove, and adjust the concentration of components. In addition, you can select from a range of packaging, QC tests, and either cGMP or non-cGMP manufacturing.

Answer Id: E11845

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Will Protein Thermal Shift™ assays work on all proteins?

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No; the assay works on most proteins we have tested (by some estimates up to 90%), but it may fail if detergents (sometimes used to solubilize membranes) are present in the buffer, if the protein does not have enough hydrophobic residues to provide a strong fluorescent signal (i.e., small proteins), or if the protein has too many exposed hydrophobic residues, resulting in high background fluorescence (might be the case for large macromolecular complexes). Each protein will require different optimal conditions; however, most proteins are successful with the first set of screens.

Answer Id: E7578

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How do protein glycosylation patterns compare between Pichia pastoris yeast, insect cells (Sf9, Sf21, HighFive) and mammalian cells?

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Answer

Pichia glycosylation consists mostly of short chain Man(10) GlcNAc residues. Compared to other yeast like S. cerevisiae which tend to hyperglycosylate, Pichia glycosylation is closer to the typical mammalian high-mannose glycosylation pattern. O-linked oligosaccharides are present but are not major components of the total soluble glycoprotein of Pichia. Standard N-linked or O-linked glycoslation amino acid consensus sequences must be present in the expressed protein in order for glycosylation to take place in Pichia.

In insect cells infected with baculovirus, the nature of N-linked glycosylation is dependent on the protein expressed, the host cell line used and the length of time of infection of cells with baculovirus. Baculovirus infection alters normal glycosylation and oligosaccharide processing characteristics of insect cells. N-linked glycosylation is generally of the high-mannose type. O-linked glycosylation is, although not identical, similar to mammalian cells depending on localization and type of protein.

We also offer the Mimic™ insect cell line. Mimic™ cells are transgenic insect cells that have been engineered to produce recombinant proteins with terminally sialylated N-glycans like those found in mammalian systems. These are identical to the SfSWT-1 cells developed by Donald L. Jarvis. This cell line is suitable for expressing recombinant glycoproteins with baculovirus and other insect expression systems, including the Bac-to-Bac™ Expression System and the InsectSelect™ system.

References for glycosylation in Pichia:
1) Grinna LS, et al. Size distribution and general structural features of N-linked oligosaccharides from the methylotrophic yeast, Pichia pastoris. Yeast. 1989 Mar-Apr;5(2):107-15.
2) Juge N, et al. Overexpression, purification, and characterization of recombinant barley alpha-amylases 1 and 2 secreted by the methylotrophic yeast Pichia pastoris. Protein Expr Purif. 1996 Sep;8(2):204-14.
3) Trimble RB, et al.Structure of oligosaccharides on Saccharomyces SUC2 invertase secreted by the methylotrophic yeast Pichia pastoris. J Biol Chem. 1991 Dec 5;266(34):22807-17.
4) Tschopp JF, et al.Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res. 1987 May 11;15(9):3859-76.

Answer Id: E3747

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What is the magnetic susceptibility for Dynabeads™ magnetic beads?

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Answer

Magnetic susceptibility is a measure of how quickly the beads will migrate to the magnet. This will depend on the iron content and the character of the iron oxide. The magnetic susceptibility given for the Dynabeads™ magnetic beads is the mass susceptibility, given either as cgs units/g or m^3/kg (the latter being an SI unit). For ferri- and ferromagnetic substances, the magnetic mass susceptibility is dependent upon the magnetic field strength (H), as the magnetization of such substances is not a linear function of H but approaches a saturation value with increasing field. For that reason, the magnetic mass susceptibility of the Dynabeads™ magnetic beads is determined by a standardized procedure under fixed conditions. The magnetic mass susceptibility given in our catalog is thus the SI unit. Conversion from Gaussian (cgs, emu) units into SI units for magnetic mass susceptibility is achieved by multiplying the Gaussian factor (emu/g or cgs/g) by 4 pi x 10^-3. The resulting unit is also called the rationalized magnetic mass susceptibility, which should be distinguished from the (SI) dimensionless magnetic susceptibility unit. In general, magnetic mass susceptibility is a measure of the force (Fz) influencing an object positioned in a nonhomogenous magnetic field. The magnetic mass susceptibility of the Dynabeads™ magnetic beads is measured by weighing a sample, and then subjecting the sample to a magnetic field of known strength. The weight (F1) is then measured, and compared to the weight of the sample when the magnetic field is turned off (F0). The susceptibility is then calculated as K x 10^-3 = [(F1-F0) x m x 0.335 x 10^6], where K is the mass susceptibility of the sample of mass m. The susceptibility is then converted to SI units.

Answer Id: E7922

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How do I decrease background autofluorescence when doing immunostaining?

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Answer

Autofluorescence is due to either the inherent fluorescent properties of natural biomolecules (e.g. lipofucsin, flavins, beta-carotene, etc.) or the reaction of aldehyde-based fixatives with various tissue components. Fixative-induced autofluorescence is higher with glutaraldehyde than with formaldehyde and is also higher when the fixation incubation time is longer or incubation temperatures are warmer.

Autofluorescence from natural pigments may be reduced by the use of picric acid, Sudan Black, or copper sulfate. Fixative-induced autofluorescence may be reduced by soaking samples in a buffered solution of either sodium borohydride, sodium cyanoborohydride, or ammonium chloride. These methods may not work consistently in every sample and may require some empirical testing.

Another method to reduce both forms of autofluorescence is to photobleach the sample prior to the use of any fluorescent probes (Neumann and Gabel (2002) J Histochem Cytochem 50:437-439.

Answer Id: E4930

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What is the Tilt Base for?

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Answer

The Tilt Base inclines the GeneAmp™ 9700 PCR System sample block by 11 degrees C, the angle required for optimal performance of the QuantStudio™ 3D Digital PCR 20K Chips.

Answer Id: E7161

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