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Do you have any specific recommendation on the type of non-stick tubes to use for diluting the Poly-A RNA Control Stock? Product FAQ

Answer

The following tubes were used in-house during product development of this Poly-A RNA Control Kit, and we have not tested any other tubes at this time:
Non-stick RNase-free 0.5 mL microfuge tubes, Ambion, Cat. No. 12350
Non-stick RNase-free 1.5 mL microfuge tubes, Ambion, Cat. No. 12450

Answer Id:: E14385

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SureSpin 632 Rotors Instruction manual Product Literature

What are the volumes of your 12x75mm, 17x100mm, and other sizes of Samco culture tubes? Product FAQ

Answer

The approximate volumes of our standard size culture/test tubes are as follows: 12 x 75 mm = 5 mL, 13 x 100 mm = 8 mL, 16 x 125 mm = 17 mL, 16 x 150 mm = 20 mL, 17 x 100 mm = 15 mL.

Answer Id:: E15924

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Smart Note: What are the top three best practices for using “sticky mats” instead of metal clamps to secure flasks, tube racks and other items during agitation on an orbital shaker? Product Literature

I have other microfuge tubes available in the lab, including FACS tubes. Can I use the PrimeFlow RNA Assay with other microfuge or FACS tubes? Product FAQ

Answer

The use of other microfuge tubes or polystyrene FACS tubes during the hybridization steps of the PrimeFlow RNA protocol may result in significant cell loss and weaker signal. The microfuge tubes provided with the PrimeFlow RNA Assay have been validated to minimize cell loss and maximize signal. We highly recommend that you only use the provided microfuge tubes. However, cells may be fixed and permeabilized in bulk using polypropylene conical tubes (e.g. Fisher Scientific Cat. No. 14-959-70C). A modified protocol for the use of polystyrene, 96-well plates is also available in Appendix 7 of the PrimeFlow RNA Assay User Manual.

Answer Id:: E14480

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Can Phasemaker Tubes be used with organic reagents other than TRIzol Reagent? Product FAQ

Answer

Phasemaker Tubes work exclusively with TRIzol Reagent (and TRIzol LS Reagent ). As other similar reagents may have different density, Phasemaker Tubes may not work properly.

Answer Id:: E15196

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Benchmark performance of Matrix 2D-barcoded cryostorage tubes Product Literature

Compatible racks for the ID Scribe Labware Product Literature

How can I use Dynabeads Protein G beads for immunoglobulin (Ig) capture? Do you have a protocol for this? Product FAQ

Answer

The procedure described below is for the isolation of Igs from a 100 µL sample containing between 0.2 µg (2 µg/mL) and 250 µg (2.5 mg/mL) Igs. The protocol can be scaled up or down as required.

Washing Procedure

1. Resuspend the Dynabeads Protein G thoroughly to obtain a homogeneous suspension. 2. Transfer the desired volume of Dynabeads Protein G to a tube at room temperature. In order to isolate Ig from a 100 µL sample, it is generally recommended to use 20-100 µL of the Dynabeads Protein G. A larger volume can be used if the sample has a high Ig concentration. 3. Place the tube on the magnet for 1 min and discard the supernatant by aspiration with a pipette while the tube remains on the magnet. 4. Remove the tube from the magnet, add 0.5 mL of a citrate-phosphate buffer*, pH 5.0, and resuspend the Dynabeads Protein G. 5. Repeat steps 3, 4 and 3.

Ig Capture Procedure

1. Add 100 µL sample containing Igs to the washed Dynabeads Protein G.
2. Incubate with gentle mixing for 40 min at room temperature. It is important to keep the Dynabeads Protein G in suspension during this step
3. Place the test tube on the magnet for 2 min and discard the supernatant.
4. Remove the test tube from the magnet and add 0.5 mL citrate-phosphate buffer, pH 5.0. (For downstream immunoprecipitation or storage of Dynabeads Protein G, 0.01_0.1% Tween-20 can be added to the buffer to prevent aggregation).
5. Wash the Dynabeads Protein G by repeating steps 3 and 4 twice.
6. Place the test tube on the magnet for 2 mins and discard the supernatant. The captured Igs are now ready to be eluted off the Dynabeads Protein G, or the Dynabeads Protein G-Ig complex can be used for immunoprecipitation.

Ig Elution Procedure

Elution of Igs is, in this example, performed by lowering the pH using 0.1 M citrate buffer (pH 2-3). The acid pH needed depends on the species and Ig subclass, but at pH 3.1 most Igs will elute.

1. Add 30 µL 0.1 M citrate (pH 2-3) to the Dynabeads Protein G-Ig complex.
2. Mix well by tilting and rotation for 2 mins.
3. Place the test tube on the magnet for 1 min and transfer the supernatant containing purified Igs to a new tube.
4. Repeat step 1, 2, and 3 in order to elute any remaining Ig. Pool the supernatants containing the pure Igs (total collected volume = 60 µL).

*Washing Buffer Recipe:

To make the citrate-phosphate buffer, pH 5, mix 4.7 g citric acid (MW=192) with 9.2 g dibasic sodium phosphate (Na2HPO4) dihydrate (MW=178) in distilled water and mix to dissolve. Bring the final volume to 1 L with more distilled water.

Please note: in the protocol we recommend using citrate-phosphate buffer pH 5.0; however, it is also possible to use other buffers like 0.1 M Na-citrate pH 5.0 or 0.1 M Na-acetate pH 5.0.

Answer Id:: E13020

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What are some general quality considerations for drug discovery assays? Product FAQ

Answer

In general, we would recommend considering the following:

Water
- Ultrapure water may be derived from water treatment systems, but the spigot or tubing attached to spigots can become contaminated and in turn can contaminate the water as it is dispensed.
- Please make sure that the membrane/filters are changed.
- Purchase the highest-grade HPLC water.
- Mineral contamination in water:
- The chelates used in TR-FRET assays are very sensitive to Fe3+ contamination. Fe3+ competes with the Tb3+ or Eu3+ for the chelate and leads to a diminished or no assay window.

Plastics
- Sequestering of either the kinase or nuclear receptor, substrate, test compound or any component used in the assay can occur with different sorts of plastics.
- Use the assay plates recommend in the protocol if there is one.
- For TR-FRET Assays, white plates of the same plastic can be substituted for the black plates and they do perform better on some instruments and for assays with small windows.
- White plates cannot be used for fluorescence polarization (FP) experiments.
- Once an assay has been established and is running smoothly, please feel free to compare the results with any assay plate that you use more regularly.

Instrument
- Optimize and test the instrument prior to performing the assay.
- Test the instrument settings using reference fluorophores or standards.
- Include a water blank of the same volume as the assay. Since a “water blank” is non-fluorescent, it provides information on instrument noise, electrical variances or other issues unrelated to the assay reagents.
- Contact the instrument manufacturer to improve optimization. Optimizing the instrument may include adjusting the gain (distance of optics from sample) and the voltage on the photomultiplier tube (PMT; the detector).

Answer Id:: E16532

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The value is not the expected value when using the Qubit Fluorometer. What could be the reason for this? Product FAQ

Answer

Please see the possibilities below:

- If you have been reading UV absorbance and your value is lower than expected, it is likely that your sample is contaminated with other molecules that absorb at 260 nm.
- The initial value that the Qubit Fluorometer shows is the concentration of the biomolecule (DNA, RNA, or protein) in the assay tube. To determine the concentration of the biomolecule in the SAMPLE, use the "Calculate Stock Concentration" or "Calculate Sample Concentration" feature on the results screen.
- Check the Qubit assay kit product manual for a list of contaminants that could interfere with the assay.
- Ensure that the lid is completely closed when reading standards and samples.
- Ensure that you have made a fresh dilution of the dye in buffer (1 µL dye to 199 µL buffer.)
- Ensure that you have labeled your tubes correctly.
- Ensure that the kit has not passed its expiration date.
- Ensure that the dye has been stored in the dark.
- Ensure that the buffer and dye are both stored at room temperature. It takes hours for a bottle of buffer at 4°C to warm to room temperature.
- Check for air bubbles in the solution. Bubbles on top are OK, but bubbles lower in the solution or at the bottom may affect the reading.
- To reduce pipetting error, dilute the sample and use a larger volume. This is especially important when pipetting concentrated or viscous solutions.

Answer Id:: E8148

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How will Dynabeads Streptavidin Beads behave in Dynabeads KilobaseBINDER Buffer? Product FAQ

Answer

The Dynabeads KilobaseBINDER Buffer is a viscous solution and therefore the beads will behave differently in this buffer than in other buffers. Be patient while resuspending the beads in the buffer and try to avoid pumping air into the tube. Flicking the tube containing the beads and buffer must be done very carefully.

If still not completely dissolved, leave it on a roller at 4 degrees C.

Answer Id:: E6162

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I am trying to assay cell proliferation with a CellTrace stain, and I am not seeing separate peaks for each cell division. How can I optimize this assay? Product FAQ

Answer

The key to good generational profiles with CellTrace reagents is starting with cells that are evenly labeled so that they have a tight coefficient of variance (CV) when run at time zero after labeling. If the peak is too broad, the generations will overlap each other and the series of peaks will become a hump. Even labeling can be achieved by starting with a uniform cell population (not a mixture of lymphocytes and granulocytes for example) as staining will be proportional to cell size. Cells are labeled rapidly, so you want to pre-dilute the dye and mix it into your cells rapidly. Be sure that the cells are not sitting in a clump in the bottom of your tube. The easiest way to do this is to make a 2x dye solution (1x = 1-10 µM) and resuspend your cells in a half volume of medium (no serum or BSA). Add the dye to the cells and invert a few times to mix. Gently agitate the cells during staining. Once the dye incubation is over (20 min, 37 degrees C), add serum or BSA (at least 1%) to scavenge any remaining unreacted dye. Spin down cells, wash 1x, and resuspend in complete medium. After a 10-20 min incubation to undergo de-esterification, cells are ready to be set up for whatever treatment you are planning. Be sure to keep a time zero control as you need to know where the first generation ran.

Answer Id:: E14848

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I am getting inaccurate results/poor reproducibility with the Qubit Protein Assay Kit. Can you offer some tips? Product FAQ

Answer

Here are possible causes and solutions:

- The kit has expired or has been stored incorrectly: When properly stored, the components of the Qubit Protein Assay Kit should be stable for at least 6 months. Upon receipt, the kit can be stored at 4 degrees C. Components A and B can be stored at ambient temperature and Components C-E can be stored at ≤4 degrees C. Protect Component A from light. High degradation of the BSA standard 2 and 3 will result in a decrease in signal and a “Standards Incorrect” error warning upon calibration. Replace the kit.
- Old calibration data was used: Best practice is to prepare fresh calibration standards at the same time as the samples to take into account any changes in assay conditions.
- Incorrect tubes were used: Use the recommended Qubit Assay Tubes (Cat. No, Q32856) or Axygen PCR-05-C tubes. Other 0.5 mL thin-walled PCR tubes may work as well, but performance is not guaranteed. Avoid opaque tubes, as these will block the light path.
- Tubes contain bubbles or particulates that are scattering the light: Pipette gently to avoid the introduction of bubbles. Spin down tubes before measuring to remove bubbles or particulates. Spin down samples to remove particulates before adding an aliquot to the Qubit working solution.
- Inaccurate pipetting: The Qubit assay will accept 1-20 µL of sample, but pipetting very low volumes, especially 1-2 µL is typically very inaccurate, especially with viscous samples. If possible, pipette at least 5 µL for more consistent results.
- The temperature of the assay is changing: Make sure that the Component B buffer is at ambient temperature before use and avoid leaving the samples in the Qubit instrument or near an exhaust fan or other heat source that would warm up the samples.
- Contamination in buffer causing high background: High buffer contamination will show up as an increase in the relative fluorescence (RFU) of the background, measured with the standard tube 1 blank, and eventually will trigger a “Standards Incorrect” error warning on calibration. Replace the kit.
- Sample buffer contains detergents: Qubit protein assay is a detergent-based assay, utilizing an environmentally sensitive dye that fluoresces in the presence of detergents; therefore, only very low concentrations of additional detergents are tolerated in the assay, as listed in Table 2 in the manual (https://tools.thermofisher.com/content/sfs/manuals/Qubit_Protein_Assay_UG.pdf).
- Sample buffer contains other components that are affecting the assay: The Qubit protein assay is generally tolerant of reducing reagents, salts, free nucleotides, amino acids, DNA, and solvents. Table 2 in the manual (https://tools.thermofisher.com/content/sfs/manuals/Qubit_Protein_Assay_UG.pdf) lists acceptable concentrations for many common contaminants. If you have a contaminant you think is affecting the quantitation, then prepare duplicate standard tubes, and spike the contaminant into one set of tubes and run them as samples. If the effect is not too substantial, then spike the buffer into the standards when performing the calibration to account for buffer composition effects.

Answer Id:: E15593

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