Product FAQ

What kind of dynamic range can I expect from Affymetrix™ miRNA 3.1 Array Plates?

Answer

Affymetrix™ miRNA 3.1 Array Plate provides a similar dynamic range to GeneChip™ miRNA 3.0 Array.

Answer Id: E14201

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Product FAQ

During Western transfer conditions using constant voltage, what would cause the current reading to drop much lower than the expected starting current?

Answer

There are three common explanations:

1) The buffer was accidentally made too dilute, which increases resistance and lowers conductivity and current. Check the transfer buffer and its reagent components, remake, or redilute.

2) The circuit is broken or impeded, as in the case of a corroded or broken electrode or malfunctioning power supply. Check the equipment.

3) There is a leak in the blot module. This is indicated by a drastic decrease in current and in buffer volume within the module.

Answer Id: E3286

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Product FAQ

How do I use the new .ps files for limiting the data output to human, mouse, or rat probe sets in Expression Console Software?

Answer

To use the new .ps files, you must download the Affymetrix™ miRNA 3.1 library analysis file package (miRNA-3_0-st-v3_analysis.library_files.20121130, or newer) from the product or support web page and place all files in the folder used for Expression Console analysis library files. Restart Expression Console Software (if open) and reload the existing study, or create a new study. When you click the “Run Analysis” button, a pop-up window will appear and provide you with a listing of available analyses. These include:
1) All probe sets for all organisms
2) Human only probe sets
3) Mouse only probe sets
4) Rat only probe sets.

All 4 of these analysis options include the choice of RMA+DABG with and without normalization.

Answer Id: E14202

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Product FAQ

Will bacterial contamination affect the growth rate of my culture?

Answer

Definitely. You can check your culture for contamination by growing culture without antibiotics. If contaminated, discard the culture. The most obvious sign of bacterial contamination is a change in the odor of a culture.

Answer Id: E3979

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Product FAQ

What is the efficiency of biotinylation? Can DNA be photobiotinylated for use as a probe?

Answer

The efficiency of photobiotinylation is variable. Efficiency depends on the purity of the sample, the intensity of the light source, and the number of repetitions. DNA will biotinylate, although not as well as RNA. DNA oligomers of 24 bp can be sufficiently photobiotinylated to work as probes, although they are too short to be used for subtraction purposes. Greater than 300 bp is usually necessary.

Answer Id: E3287

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Product FAQ

Where are Human Corneal Epithelial Cells (HCEC) isolated from?

Answer

HCECs are isolated from normal human corneal-scleral tissue. Trimmed limbal regions are enzymatically digested, and released epithelial cells are expanded. HCECs are cryopreserved using a controlled-rate freezer at the end of the secondary culture (p1) in a cryopreservation reagent containing 10% dimethyl sulfoxide (DMSO).

Answer Id: E11979

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Product FAQ

Do we have to use any Affymetrix™ Reagents with our assay?

Answer

GeneChip™ Hybridization Control Kit 30 reactions
GeneTitan™ Hybridization, Wash, and Stain Kit for miRNA Array Plates 96 reactions
GeneTitan™ Hybridization Module for miRNA Plates 96 reactions
Affymetrix™ FlashTag™ Biotin HSR RNA Labeling Kit 10 reactions
Affymetrix™ FlashTag™ Biotin HSR RNA Labeling Kit 30 reactions

Answer Id: E14203

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Product FAQ

Are the MagJET Plant Kits compatible with sample disruption using automated homogenizers?

Answer

MagJET Plant Kits are compatible with homogenization using liquid nitrogen, as well as automated homogenizers.

Answer Id: E8860

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Product FAQ

What level of purification do I need for my DNA sequencing primers?

Answer

We recommend using HPLC-purified primers for DNA sequencing. This will ensure the presence of full-length, highly purified primers that will minimize cycle sequencing noise and provides longer sequencing reads.

Answer Id: E15324

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Product FAQ

Can I use other buffer systems from other companies when using eBioscience™ phosphorylation-specific antibodies?

Answer

In general, you can use other companies’ buffer systems provided that their buffers are of a similar composition as the buffers recommended. Be aware that results will vary depending upon the buffer system used. eBioscience™ antibodies have been optimized for use in eBioscience™ ™ buffers and we have not tested all of our antibodies in other buffer systems. Thus, we highly recommend using our optimized protocol and buffer systems.

Answer Id: E14467

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Product FAQ

Will a low cell-number innoculum reduce the growth rate of my culture?

Answer

The ability to grow from low density inoculation varies from individual cell line to cell line. Some, such as insect cell lines, can be greatly affected when passaged to too low a density. Such cells may depend on factors which they secrete themselves to maintain their growth. Passaging to too-low a density deprives them of these factors for an extended amount of time, owing to both the dilution of their spent medium and the low cell numbers secreting the factors. Such passaging will often result in an extended lag phase in which the cells show little or no growth, before finally picking up. These cells generally grow best when passaged 1:3 to 1:5, and with carryover of spent (conditioned) medium with the passage.

Other cell lines can be passaged to extremely low densities without much trouble. Hybridomas and other fast-growing cells can often be split 1:20 and show no apparent lag phase.

What cultures require most is consistency. Once a pattern of passaging is established, variations from the norm will have an effect, whether innoculating from or at a lower than normal density, or allowing the cells to stay at confluency for an extended amount of time.

Answer Id: E3981

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Product FAQ

Can you obtain a better representation of complete cDNAs if you use the random primers or oligo dT primers in the first-strand cDNA synthesis step for cDNA library construction?

Answer

First-strand cDNA synthesis is most commonly primed using oligo(dT) or modifications of this sequence (such as primer-adapters) that bind to the poly(A) tail of mRNA. This priming method offers two major advantages: only poly(A)+ RNA is copied, and most cDNA clones begin at the 3’ terminus of the mRNA.

At the same time, oligo(dT) priming has certain limitations: some cDNA clones may not be full length (due to RNA secondary structure or pausing by reverse transcriptase) and poly(A)- mRNA cannot be copied.

An alternative method is to use random hexamers, which, in theory, are capable of binding and priming throughout virtually any RNA template. Random hexamers, which may be used either by themselves or in combination with oligo(dT), have been instrumental in producing cDNAs containing more 5’ information than those primed with oligo(dT) alone. In addition, random hexamers can be used to generate cDNA libraries from poly(A)- mRNA and single-stranded viral RNAs.

Answer Id: E3288

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Product FAQ

Sometimes my molecular weight (MW) markers are no longer visible after my detection is completed with an iBlot™ Western Detection Kit. Does this mean my proteins are also gone?

Answer

No. Molecular weight markers are usually pre-stained and more highly charged than typical cellular proteins. Applying an electrical field in the western detection procedure can drive the MW markers right through the membrane onto the bottom stack.

Cellular proteins, however, are less charged than the molecular weight markers, and at the detection stage they no longer contain any SDS. The result is that the cellular proteins stay immobilized on the membrane during western detection using the iBlot™ system.

Answer Id: E11322

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Product FAQ

How many times can I passage Human Corneal Epithelial Cells (HCEC)?

Answer

HCECs are performance-tested and guaranteed to reach greater than 11 population doublings after thaw. Generally, this requires 3-4 passages when HCECs are seeded at 5,000 cells/cm2.

Answer Id: E11980

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Product FAQ

Is the Command Console web server hosted by customers or by your company?

Answer

The Command Console web server is hosted by the customer.

Answer Id: E14220

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