What is the difference between the Novex™ TBE-Urea Sample Buffer and the Novex™ Prep TBE-Urea Sample Buffer?

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Answer

We provide two optimized TBE-urea sample loading buffers: the Novex™ TBE-Urea Sample Buffer contains two tracking dyes, while the Novex™ Prep TBE-Urea Sample Buffer contains no tracking dyes. The first is used for analytical runs, and the prep sample buffer without marker dyes is used when UV shadowing will be used prior to cutting out the band or when the marker dyes may interfere with the oligonucleotide bands.

Answer Id: E8000

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What is the difference between Gibco™ Neurobasal™ Medium and Gibco™ Neurobasal™-A Medium?

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Neurobasal Medium is optimized for prenatal and fetal neurons. Neurobasal-A Medium is optimized for growing postnatal and adult brain neurons. These two media differ only in osmolality.

Answer Id: E12012

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Should Enterokinase be resuspended in a buffer containing 50% glycerol to protect the protein from freeze/thaw cycles?

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Freeze thaw has a minimal effect on the activity of Enterokinase. The addition of glycerol is not necessary but can make handling of the enzyme easier.

Answer Id: E3522

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Do your Zymogram gels have a stacking gel?

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Yes, our Zymogram gels do contain a 4% stacking gel that is ~8 to 9mm long.

Answer Id: E10729

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What is the difference in composition between the Novex™ 6% TBE Gels and the Novex™ 6% DNA Retardation Gels?

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Answer

The Novex™ 6% DNA Retardation Gels contain 0.5X TBE. Both gels will work for gel retardation; however, the 1X TBE in the Novex™ 6% TBE Gels have a higher ionic environment, which may affect DNA-protein interactions. The 0.5X TBE used in the Novex™ 6% DNA Retardation Gels usually works better, as it offers good fragment separation in electrophoresis yet has an ionic strength low enough to promote DNA-protein interactions.

Answer Id: E8001

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Which supplement can I add to Neurobasal™ Medium to culture my neural cells?

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Answer

As a general guideline, we recommend using Gibco™ B-27™ Supplement to culture neural stem cells, hippocampal, and other CNS neurons.

- For neuroblastomas, or post-mitotic neurons from both PNS and CNS, Gibco™ N-2 Supplement can be used.
- For primary glial cells (astrocytes) or tumor cell lines of astrocytic phenotype (astrocytes and gliomas), or oligodendrocytes, Gibco™ G-5 Supplement can be used.

Answer Id: E12013

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I’m trying to propagate my Gateway™ destination vector and am not seeing any colonies. What should I do?

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Answer

Check the genotype of the cell strain you are using. Our Gateway™ destination vectors typically contain a ccdB cassette, which, if uninterrupted, will inhibit E. coli growth. Therefore, un-cloned vectors should be propagated in a ccdB survival cell strain, such as our ccdB Survival™ 2 T1R competent cells.

Answer Id: E6731

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Will DTT, Triton™ X-100 detergent, Tween™ 20 detergent, Thesit, calcium chloride, sodium chloride, or SDS affect the efficiency of Enterokinase (EKMax™) enzyme cleavage?

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Answer

Enterokinase is active in buffers containing up to 1 mM DTT, 0.1% Triton™ X-100 detergent, 0.1% Tween™ 20 detergent, and 0.1% Thesit. It is recommended to have 10mM Tris pH 8.0 and 10 mM calcium chloride in the buffer. Enterokinase is inhibited by sodium chloride and SDS.

Answer Id: E3523

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Do your Zymogram gels contain the gelatin or casein substrate in the stacking portion?

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Answer

Our Zymogram gels do not contain the substrate (gelatin or casein) in the stacking gel although a small amount might mix in during casting of the gel.

Answer Id: E10730

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How do I change the barcoding set used to analyze a next-generation sequencing run?

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Answer

Follow these steps to change the DNA barcode ID:

- Open a run reports listing in the “Data,” “Completed Runs & Reports” tab.
- In the list view, click the “Edit” button on the right side of the run listing. The Edit Run window opens.
- Click the barcode menu pull-down and choose the correct barcode set. If you have custom barcode sets, your list is different. Click the “Save” button (in the bottom right corner).
- You can now reanalyze the run from the “Data,” “Completed Runs & Reports” tab.

Answer Id: E10475

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What is a shift assay? What is a supershift assay?

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Answer

A shift assay is a DNA-binding assay using nondenaturing PAGE. It provides a simple, rapid, and extremely sensitive method for detecting sequence-specific DNA-binding proteins. Proteins bind specifically to an end-labeled DNA fragment corresponding to the individual protein-DNA complexes. You can use the assay to test binding of purified proteins or of uncharacterized factors in crude extracts. This assay also permits quantitative determination of the affinity, abundance, association rate constants, dissociation rate constants, and binding specificity of DNA-binding proteins.

A supershift assay is a variation of the mobility shift DNA-binding assay that uses antibodies to identify proteins present in the protein-DNA complex.Addition of a specific antibody to a binding reaction can have one of several effects. If the protein recognized by the antibody is not involved in complex formation, addition of the antibody should have no effect. If the protein that forms the complex is recognized by the antibody, the antibody can either block complex formation or it can form an antibody-protein-DNA ternary complex and thereby specifically result in a further reduction in the mobility of the protein-DNA complex (a supershift). Results may be different depending upon whether the antibody is added before or after the protein binds DNA (particularly if there are epitopes on the DNA binding surface of the protein).

Answer Id: E8002

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Why is the color of Neurobasal™ Medium without Phenol red yellow?

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Answer

The yellow color comes from vitamin components.

Answer Id: E12014

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How should I store my lentivirus?

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Answer

Lentivirus should be aliquoted immediately after production and stored at -80 degrees C. Lentivirus can lose up to 5% of titer with each freeze/thaw. When stored properly, viral stocks should be suitable for use for up to one year. After long-term storage, we recommend re-titering the viral stocks before use.

Answer Id: E6387

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Are Thermo Scientific™ ABgene plastics sterile?

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Answer

Thermo Scientific™ Abgene plastics are not typically sterilized as their entire production process, from molding to final packaging, is carried out in a Class 100,000 clean room under ISO 9001 guidelines. All Thermo Scientific™ PCR plastics are certified free from RNase, DNase and human DNA. In contrast, during typical non-clean room production, plastics are exposed to many contaminants including dust, bacterial cells, and DNA. The plastics are then sterilized to kill bacteria and inactivate RNases and DNases, but sterilization does not remove dust or DNA contamination. The dust particles left behind can inhibit PCR, and the damaged DNA fragments can still act as templates, leading to non-specific amplification.

Answer Id: E8807

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I’m only getting white colonies, but none of the clones have an insert. What can I do?

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Answer

Here are a few suggestions:

- Small fragments/linkers are cloning in to your vector instead of your insert; to correct this, gel-purify the insert before ligation
- Ensure that the correct concentrations of X-gal and/or IPTG (if vector contains the lacIq marker) are used
- If spreading X-gal and/or IPTG on your plate, allow sufficient time for the reagents to diffuse into the plate
- Incubate your plate for a longer period to ensure full color development

Answer Id: E6732

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