Why is the background higher on low-percentage acrylamide Tris-Glycine gels after staining with Colloidal Blue?

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Answer

Background is generally higher in gels less than 10% in acrylamide concentration due to penetration and trapping of colloids within the large pores of the low-percentage acrylamide gels. Excess background may be removed by incubating in 25% methanol solution until a clear background is obtained. Be aware that dye will also be partially removed from the bands and prolonged incubation in >25% methanol will result in a complete destaining of protein bands and background.

Answer Id: E11249

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Can any type of cell incorporate EdU?

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No. The cell must possess a pyrimidine salvage pathway—without this pathway, EdU does not become phosphorylated to allow incorporation into replicating DNA.

Answer Id: E10795

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Can the bacluovirus infect mammalian cells? How about Drosophila cells?

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Answer

Yes, baculovirus can infect mammalian cells, although only at very high titers. Baculovirus works best in liver cells. However, there is no danger of cross-contamination unless the cells are directly infected with the high-titer stocks. Bacuolvirus can infect Drosophila cells; however, it will not replicate in these cells. The promoters used to drive expression of your gene in a typical baculovirus system are both late promoters and require earlier proteins from the baculovirus genome. Thus, they will not work in S2 cells since the early proteins are not made.

Answer Id: E9433

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How are bead density and volume measured?

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Helium pycnometry is a method for determining density and volume by measuring the pressure change of helium in a calibrated volume. The technique measures sample volume, from which density can be derived automatically if sample weight has been entered.

Answer Id: E6206

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Do you sell a strain for production of recombinant bacmids?

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Yes, we would recommend using our MAX Efficiency™ DH10Bac™ competent cells. The DH10Bac™ E. coli strain contains a bacmid, which can recombine with our donor plasmid, pFastBac, to create an expression bacmid. These cells are a part of our Bac-to-Bac™ Baculovirus Expression System.

Answer Id: E6707

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I used the Thermo Scientific™ Pierce™ Silver Stain for Mass Spectrometry and my bands are very faint or not visible at all. What should I do?

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Answer

Here are possible causes and solutions:

- Insufficient development time. Develop gel for more than 5 minutes or add freshly prepared Developer Working Solution.
- Minimal or no protein present in sample. Check protein concentration in the original sample.
- Improper solution preparation or skipped steps. Check solution preparation and follow procedure.
- Excessive water wash before development step. Wash gel three times for 10 minutes each to completely remove the previous solutions and redo the staining procedure starting at step 2. Do not over wash prior to incubation in the developer.

Answer Id: E11250

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What is the difference between absolute quantification (AQ) and relative quantification (RQ)? How do I choose which method to use?

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Absolute quantification will quantitate unknowns based on a known quantity. It involves the creation of a standard curve from a target of known quantity (i.e., copy number). Unknowns can then be compared to the standard curve and a value can be extrapolated. Absolute quantification is useful for quantitating copy number of a certain target in DNA or RNA samples. The result usually is a number followed by a unit, such as copy number and ng, etc.

Relative quantification can quantitate a fold difference between samples. It involves the comparison of one sample to another sample (calibrator) of significance. For example, in a drug treatment study you could compare a treated to an untreated sample. The quantity of the calibrator is not known and cannot be measured absolutely. Therefore the calibrator (untreated sample) and samples (treated samples) are normalized to an endogenous control (a gene that is consistently expressed among the samples) and then compared to each other to get a fold difference. Relative quantification is useful for quantitating messenger RNA levels. Since the result is a fold change or ratio, it is not followed by a unit.

The method that you choose will depend on the type of data you need from your experiment. You can find more information here (https://www.thermofisher.com/us/en/home/life-science/pcr/real-time-pcr/qpcr-education/absolute-vs-relative-quantification-for-qpcr.html) as well.

Answer Id: E7494

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What does DE3 mean?

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Answer

The DE3 designation means the strains contain the lambda DE3 lysogen that carries the gene for T7 RNA polymerase under control of the lacUV5 promoter. This promoter is regulated by the endogenous E. coli lacI protein and is induced with IPTG. IPTG is required to induce expression of the T7 RNA polymerase. The DE3 lambda derivative also contains the immunity region of phage 21.

Answer Id: E9728

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Can any type of cell incorporate EU?

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Answer

No. The cell must possess a pyrimidine salvage pathway; without this pathway, EU does not become phosphorylated to allow incorporation during RNA synthesis.

Answer Id: E10796

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Can a P3 viral stock be used to generate more viruses? How about a P4 or P5 stock?

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Answer

Yes, the same protocol used to make your P2 viral stock can be used to make a P3, P4, or P5 viral stock. We don’t recommend making the stock higher than P5, as more defective interfering particles will be produced and a decrease in protein expression level will occur.

Answer Id: E9434

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What happened to the PanVera™ FlAsH EDT2 Labeling Kit, Cat. No. P3050?

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The FlAsH-EDT2 reagent has been renamed Lumio™ Green Labeling Reagent. It is available in the Lumio™ Green In-Cell Labeling Kit, Cat. No. 12589-057. While the kit is designed for staining live cells expressing proteins containing the Lumio™ fusion tag, you may substitute the Lumio™ Green labeling reagent directly into your existing protein labeling protocol. The kit contains the Lumio™ Green reagent and Disperse Blue 3 for background suppression. We also have available the Lumio™ Red In-Cell Labeling Kit, Cat. No. 12589-040.

Answer Id: E4401

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When should “T” buffer be used instead of “R” buffer with the Neon™ system?

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Answer

You need T buffer instead of the standard R buffer for primary T and B cells. These cells are smaller than regular cell types and require higher voltage for successful electroporation. If you use R buffer and apply high voltage, you will see sparks.

With R buffer, voltage over 1800V generally generates sparks regardless of cell number and other condition. The maximum voltage for R buffer is around 1900V. In order to apply higher voltage, you need a buffer less conductive than R buffer, which is the T buffer.

Answer Id: E5529

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Is it always necessary to use the transport locks on the KingFisher™ Flex instrument?

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Answer

The transport locks are only necessary when relocating the instrument.

Answer Id: E10355

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What is the refractive index of Dynabeads™ magnetic beads?

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Answer

Unfortunately, we don't have any data regarding refractive index of either coated or uncoated magnetic beads, but the refractive index for polystyrene-divinylbenzene beads (beads in the earlier phases of process) is approximately 1.5-1.6.

Answer Id: E6207

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Can I use TOP10F’ competent cells for transformation of my TOPO™ vector that contains the ccdB gene?

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Answer

Strains that contain an F plasmid, such as TOP10F’, are not recommended for transformation and selection of recombinant clones in any TOPO™ vector containing the ccdB gene. While the F plasmid does encode the CcdA protein, which acts as an inhibitor of the CcdB gyrase-toxin protein, the half-life of the CcdA protein is shorter than that of the CcdB protein. Overexpression of the CcdB protein causes cell death when its action is not prevented by sufficient CcdA.

Answer Id: E6708

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