Product FAQ

What is the GeneChip™ Command Console™ Software?

Answer

The GeneChip™ Command Console™ Software is the next generation of Affymetrix™ core software and replaces the GCOS software suite. Command Console Software provides instrument control, sample registration, and data management capabilities. It supports the microarray workflow from sample registration through CEL file generation.

Answer Id: E14210

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What is the doubling time of Drosophila S2 cells?

Answer

When grown at the optimal temperature of 27-28ºC, Drosophila S2 cells grow very quickly, with an approximate 14-16 hr doubling time. The cells can also be grown at 22-25ºC with doubling time of 24hrs. Humidity is an issue, so a shallow pan of water can be left in the incubator, but the incubator does not need to be turned on.

Growth medium is also a factor in doubling time - the doubling times listed here are for Sf900 II medium.

The cells can reach maximum densities of 1 to 2.5 x 10E7 cells/ml in shake flask cultures.

Answer Id: E3312

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

When was Command Console Software launched?

Answer

Command Console Software was first launched in October 2007.

Answer Id: E14211

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

I am using a quantitative DNA standard and see little difference between the bands when they are ethidium bromide stained. How can this occur and what can I do?

Answer

Here are a few suggestions:

(1) Excess volume loaded on gel. The smallest loading volume will result in the greatest difference in band thickness between different masses. Basically overloading the ladders gives saturated bands which do not show any variation in intensity. It is best to load all samples in the same volume.

(2) Sample loaded in larger or smaller volume than standard. Load equal volumes of sample and standard DNA for accurate comparison.

(3) Ethidium bromide (EtBr) staining after electrophoresis. Include ethidium bromide in the gel rather than staining after electrophoresis to increase the difference in band intensity.

(4) EtBr was in the gel, but not the running buffer. This results in the absence of EtBr in the lower half of the gel after electrophoresis and therefore differential fluorescence between bands in the upper half of the gel and the lower half of the gel.

Answer Id: E3993

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Will Invitrogen™ Cre Recombinase work with any Lox sites?

Answer

We have only tested loxP and loxH sites. However, there are many mutant forms of lox out there, including LoxB, L and R. LoxP consists of two 13bp inverted repeats flanked by an 8bp central sequence. LoxH is a mutant derivative of loxP, which has mutations at the 3,6,9 nucleotide of the left repeat, creating three mismatches between the two repeats. Here are the sequences:

LoxP: ATA ACT TCG TAT AGC ATA CAT TAT ACG AAG TTA
LoxH: ATT ACC TCA TAT AGC ATA CAT TAT ACG AAG TTA

Here are two references that have studied Cre recombinase: Araki et al. (1999, Cell Mol Biol 45: 737-750) suggests that lox71 and lox66 sites work with Cre recombinase and Abremski et al. (1986, J Biol Chem 261: 391-396) suggests that lox501 sites show a 10-fold reduction in recombination activity compared to a loxP site.

Answer Id: E3316

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What is the doubling time for Human Keratinodyte?

Answer

Each lot is tested for doubling time and the results are listed on the COA.

Answer Id: E11988

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What modules are included with Command Console Software?

Answer

Command Console Software is composed of four main modules: the Command Console™ Portal registers arrays and samples and manages data; the Command Console™ Scan Control provides instrument control for the GeneChip™ Scanner 3000; the Command Console™ Fluidics Control provides instrument control for the GeneChip™ Fluidics Station 450; and the Command Console™ Viewer provides features for viewing images and grid results, manual gridding tools, and production of probe cell intensities (CEL files). Version 2.0 introduced GeneTitan™ Control, which provides instrument control for the GeneTitan™ Instrument. When used with the GeneTitan Instrument, GeneTitan Control replaces Scan and Fluidics control modules.

Answer Id: E14212

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Are Thermo Scientific™ DNA ladders composed of linear or circular/supercoiled DNA?

Answer

Thermo Scientific™ DNA ladders contain linear dsDNA fragments.

Answer Id: E8869

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What is the control in the BigDye™ Terminator kit?

Answer

The control is pGEM™-3Zf(+) , plasmid DNA which serves as a double-stranded control

Answer Id: E15333

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

For the PrimeFlow™ RNA Assay, what controls do I need?

Answer

1. To ensure proper assay performance, please use the Positive Control Probe Sets in every experiment. Please refer to Appendix A5 for specific cell types and recommended positive control genes.
2. The PrimeFlow™ Compensation Kit (included with the PrimeFlow™ RNA Assay, Cat. No. 88-18005) should be used for single-color compensation controls for the RNA detection channels.
3. Single-color compensation controls for fluorochrome-conjugated antibodies must also be included in each experiment. The UltraComp eBeads™ microspheres included in the compensation kit may also be used for single-color compensation controls for any experimental antibodies being used in the experiment.
4. In addition to single-color compensation controls, Fluorescence-Minus-One (FMO) controls are highly recommended. The FMO control is a sample that contains all but one of the fluorochromes being used in the experiment. As with single-color controls, there should be an FMO control for every fluorochrome being used in the experiment. FMO controls facilitate assessment of background on gated events and allow fine-tuning of compensation for optimal performance.
5. Negative controls are highly recommended. Samples with the target probe omitted or samples with a target probe not expressed in the cells of interest (such as the bacterial gene DapB) should be used. Additionally, biological or experimental controls comprised of or containing cells known to be negative for the gene of interest (e.g., unstimulated or uninfected) are also recommended to confirm specificity of the target probes.

Answer Id: E14476

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

How can I improve transfection efficiency with cationic lipids?

Answer

1. Select the cationic lipid reagent that is likely to result in highest transfection efficiency for your cell type. Please refer to the Transfection Reagent Selection Guide (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-reagent-application-table.html) to make the right choice.
2. Optimize the cationic lipid reagent and DNA amounts. The most important parameter after the condition of the cells is the ratio of lipid to DNA.
3. Do not use serum during complex formation. Serum may contain components that could interfere with complex formation. We recommend using Opti-MEM™ I Reduced-Serum Medium for optimal complex formation. However, serum-free DMEM or serum-free RPMI 1640 Medium can be used, keeping in mind that the efficiency of complex formation may not be as high as with Opti-MEM™ I Reduced-Serum Medium.
4. Do not use antibiotics, EDTA, citrate, phosphate, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the medium used to prepare the DNA-cationic lipid reagent complexes.
5. Cell density should be from 50% to 80% confluency at the time of transfection (for Lipofectamine™ 2000, we recommend >90% confluency). Cells should be in the mid-log growth phase. For better consistency of results between transfection experiments, it would be best to accurately count your cells with a hemocytometer or with the Countess™ II FL Automated Cell Counter (Cat. No. AMQAF1000).
6. Make sure the promoter-enhancer of the transfected DNA is compatible with the target cell type.
7. Do not use cationic lipid reagent that has been frozen or stored in a section of the refrigerator where the temperature is below 4 degrees C.
8. Include a positive control for the transfection assay (for example, Cat. No. A14146 for plasmid DNA transfection and Cat. No. 14750100 for siRNA transfection).

Also, please take a look at the tips outlined here (https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection-support/troubleshooting-transfection-experiments.html).

Answer Id: E3994

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Are there common restriction sites that can be used to excise a gene out of a Gateway™ plasmid?

Answer

The core region of the att sites contains the recognition sequence for the restriction enzyme BsrGI. Provided there are no BsrGI sites in the insert, this enzyme can be used to excise the full gene from most Gateway™ plasmids. The BsrGI recognition site is 5'-TGTACA and is found in both att sites flanking the insertion site.
If a different restriction site is desired, the appropriate sequence should be incorporated into your insert by PCR.

Answer Id: E3317

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

I am getting high background after Alexa Fluor™ 790 and 680 detection. Can you please offer some tips?

Answer

Here are possible causes and solutions:

- Insufficient blocking or non-specific binding: Try a different blocking reagent or increase the concentration of the blocking reagent. We generally obtain good results with 2% casein, 5% non-fat dry milk, or 1/2X fish serum.
- Membrane was blocked with BSA: Do not use BSA-containing solutions for blocking or incubating Alexa Fluor™ 680 and 790 conjugates. For primary antibodies that are incompatible with casein or milk (e.g., many anti-phosphoprotein antibodies), use fish serum or use a 0.5% BSA-containing solution for primary antibody incubation only and then switch to 2% casein or 5% non-fat milk for all other incubation steps.
- Membrane is contaminated: Use only clean, new membranes. Wear clean gloves at all times and use forceps when handling membranes.
- Higher intrinsic background with PVDF membranes: Switch to nitrocellulose membranes.
- Nitrocellulose membrane not completely wetted: Follow instructions for pre-wetting the membrane.
- Insufficient washing: Follow recommended number of washes. In some cases, it may be necessary to increase the number or duration of washes.
- Concentration of primary and/or secondary antibody is too high: Determine optimal antibody concentration by performing a dot blot and dilute antibody as necessary.

Answer Id: E11331

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

I see large, flat pancake-looking cells in my Human Keratinodyte culture. What are they?

Answer

These are senescent cells, and this is normal for adult keratinocyte culture. You always have a population of cells that are old and no longer proliferate. However younger cells, which are small in size, should keep proliferating. When a culture gets older, you see more and more large cells, and the culture will eventually stop growing. With the right care and attention, the culture should yield at least 25 population doublings.

Answer Id: E11989

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Are the Affymetrix™ Analysis Data Model (AADM) and Data Mining Tool (DMT) supported by Command Console Software?

Answer

AADM is a relational database model and is not supported by Command Console Software. Customers can access the Command Console indexer to get query capability. DMT is not supported by Command Console. Analysis capabilities similar to those in DMT are offered by various Affymetrix™ packages and by third-party GeneChip-Compatible (http://www.thermofisher.com/us/en/home/life-science/microarray-analysis/microarray-analysis-partners-programs/genechip-compatible-software-providers.html) packages.

Answer Id: E14229

Was this answer helpful?

Yes
No
Thank you for your response