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Regulatory requirements vary by product. Please see product-specific literature to determine product use.

What shoud I do if an alert "No Connect FTP Server" appears on the Ion PGM touchscreen? Product FAQ

Answer

The Ion PGM System and Torrent Server may not be connected. Please shut down the system and server and reboot them. The server may enter a system check after the reboot. Since the system check will take 3-4 hours, you can avoid it by pressing the 'c' button on the keyboard.

If there is a reason you cannot reboot the system, you can run the Ion PGM System without connection to the server. The runs will be saved inside the system. For 200 bp sequencing, this many runs can be saved inside the system:
314 chip: 40 runs
316 chip: 6 runs
318 chip: 5 runs

The data will automatically transfer to the server after connection.

Answer Id:: EJP01167

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What should I do if an error message "calibration FAILED" appears during initialization of my Ion PGM System? Product FAQ

Answer

Open the chip clamp and check for any leaks. If you see solution leaking, please remove the chip and clear the solution with a Kimwipe. DO NOT wipe or scrub as the socket could be damaged. Clean the socket by gently touching it with a damp Kimwipe. Make sure the socket is dry and replace the chip. Press the 'Calibration' button to recalibrate the chip. If it fails again, change the chip to a different one. Also, if you are using an Ion 316 Chip v2 on a v3.4 or older instrument, the instrument will not recognize the chip.

Answer Id:: EJP01171

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What shoud I do if R1, R2, R3, and/or R4 fails at Raw Traces and the screen turns red during the last step of initialization of my Ion PGM System? Product FAQ

Answer

The pH of the nucleotides may be out of range or there may have been a minor problem during measurement. Press the 'Start' button and restart the measurement. If it passes, continue to start the run. If an error appears again, please note the pH of R1, R2, R3, R4, as well as the error message, and contact Technical Support.

Answer Id:: EJP01165

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What should I do if the Recovery Solution does not come out from Ion OneTouch DL? Product FAQ

Answer

The Ion OneTouch DL cannot be repaired. However, you may order an Ion OneTouch DL Upgrade Kit and exchange some parts which may fix the problem.

Answer Id:: EJP01180

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What is the picture on the touchscreen during an Ion Proton run that looks like a bubble moving? Product FAQ

Answer

The touchscreen shows the voltage measured on the chip and there will be some contrast according the signal intensity which may look like bubbles.

Answer Id:: EJP01173

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Which Dynabeads magnetic bead size should I choose for isolation of cell organelles? Product FAQ

Answer

If the sample is very viscous, a large bead is preferred (4.5 µm), which has a higher magnetic content and moves more easily to the magnet in very viscous samples. If high binding capacity is preferred, choose the 2.8 µm bead.

Answer Id:: E12136

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Does Dynabeads Human CD3/CD28 also bind CTLA4 on T cells? Product FAQ

Answer

No, it does not. It is a specific anti-CD28 antibody.

Answer Id:: E12153

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Will the TCR-CD3 complex internalize? Product FAQ

Answer

In nature there is a constitutive internalization and re-entry of the TCR-CD3 complex to the cell surface. When Dynabeads magnetic beads bind to CD3/CD28 on the cells in culture, the TCR-CD3 complex is internalized and then degraded. When this happens the beads do not have any target on the surface of the cells to bind to and therefore the beads will be released into the medium. The bead-cell clusters last for about 3-4 days until the beads fall off the cells.

The T-cell receptor (TCR-CD3) complex consists of several different subunits where the variable immunoglobulin domains of TCR (V) bind to the ligand. The cytoplasmic tails of the CD3 subunits and TCR interact with cytosolic signaling proteins. In this cytosolic part of the complex several types of internalization signals are located.

Constitutive internalization (without ligand binding): From the literature we understand that approximately 20 tyrosine-based internalization motifs and 2 di-leucine motifs will constitutively remove the TCR- CD3 complex from the plasma membrane without ligand binding. The recycling time is approximately 100 minutes (from plasma membrane to early endosomes and back to plasma membrane), and after 10 such cycles the TCR- CD3 complex is targeted for degradation in lysosomes.

Induced internalization (with ligand binding): The ligand-dependent internalization of the TCR-CD3 complex is a ubiquitin-dependent internalization process. Upon ligand binding, ubiquitin is coupled to a certain amino acid in the cytosolic tail of the complex, which results in a clathrin-dependent internalization of the complex followed by lysosomal degradation (the internalization reaches plateau around 2 hours (Nature 375:148 (1995)). When the beads bind to CD3-CD28, the complex will actively be internalized and degraded. When this happens the beads do not have any target on the surface of the cells to bind to and therefore the beads will be released into the medium. If the beads are not removed from the medium they can re-bind to the TCR- CD3 complex when they re-appear at the plasma membrane.

Answer Id:: E12165

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How can I characterize neural stem cells (NSCs)? Product FAQ

Answer

NSCs are generally characterized by their ability to form neurospheres when plated at cloning density (Nat Methods 2:333 (2005)). NSCs can also be characterized by (1) RT-PCR of Sox1, Sox2, and Nestin or (2) immunohistochemical staining for nestin, Pax6, Sox2, and Ki67.

Answer Id:: E12190

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Why does my recombinant protein not have the activity claimed by the product materials? Product FAQ

Answer

If you carried out a test as described in our product insert and did not see any response, this could be due to several possibilities listed below:

- Protein was not reconstituted according to the instructions.
- The reconstituted protein is too old or protein might have precipitated. We recommend using the reconstituted protein within 3-6 months after reconstitution.
- Carrier protein was not added for proteins reconstituted at a concentration is less than 0.1 mg/mL. Working solutions at <0.1 mg/mL should be used immediately; we do not recommend long-term storage of solutions at this concentration.
- The protein solution was exposed to multiple freeze/thaw cycles or was exposed to high temperature.
- Proteins were handled in the wrong types of vessels (some proteins are very sticky to certain plastics).

Answer Id:: E12201

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I don't have a filter optimized for visualizing Qdot nanocrystals. Can I visualize them using a standard filter? Product FAQ

Answer

Yes, you can visualize Qdot nanocrystals using a standard filter; they will excite at any wavelength below their emission. Keep in mind that the lower the excitation value the brighter the Qdot nanocrystal fluorescence output.

Answer Id:: E12226

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My Qdot nanocrystals were brightly fluorescent before I mounted my samples; now I'm seeing a loss of fluorescence. Why is this happening? Product FAQ

Answer

Appropriate mounting media selection is very important to retain the fluorescence of Qdot nanocrystals. In our studies, Qdot nanocrystals work best with the following mountants:

HistoMount medium (Cat No. 00-8030); best for long term archiving
Cytoseal 60 Mountant
Clarion Mountant
Most polyvinyl alcohol-based mountants (limited storage time, less than weeks)
Water-based mountants (limited storage time, less than week)
Up to 50% glycerol (limited storage time, less than week)
Note: We do not recommend using ProLong mounting media with Qdot nanocrystals as it will quench their fluorescence.

Answer Id:: E12238

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I sonicated my carboxylate-modified latex microspheres as recommended in the protocol, and I saw foaming in the solution. Should I be concerned? Product FAQ

Answer

The foaming is from Tween 20, which is present in the stock solution to help prevent aggregation. It is normal to see bubbles from this reagent.

Answer Id:: E12258

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How much Trypan Blue solution should I add to my cell suspension before counting? Product FAQ

Answer

We recommend adding 0.1 mL of 0.4% Trypan Blue stock solution to 0.1 mL of cells. However, depending upon the sample and instrumentation, the ratio of 0.4% Trypan Blue solution to the volume of cells can be varied from 1: 10 to 1:1 (v/v) so that the final concentration of Trypan Blue is 0.04% - 0.2%.

Answer Id:: E12265

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We have the Countess II and Countess II FL instruments in our lab. What is the difference between the light source adjustment before the count and the cell brightness/intensity adjustment after the count? Product FAQ

Answer

The light source adjustment controls the LED intensity, camera gain, and exposure time, and it is used for adjusting the image brightness and contrast before you capture the image. Once the cells are inserted, there will be a bar on the right hand side to adjust the light source intensity. Often it helps to turn it down so that the background is slightly darker, which allows better contrast between live and dead cells. Optimal light intensity in brightfield is usually around 10-20 if there are no light cubes in the instrument, and slightly higher around 20-30 if there are light cubes present, but this could vary depending on the sample.

Once the image is captured, you can also adjust the range of cell size and cell brightness that the counting algorithm will count. This doesn't change the actual brightness of the image or how it looks, but determines which cells will be counted. Once you press “Capture” to count the cells, you can hit the “Adjust” button and make sure that the size, brightness, and circularity gates are all maximized by maximizing the slider bars to include all of the cells in the count. When you go into the “Adjust” screen, you can adjust the size, brightness, and circularity for both live and dead cells, so make sure that you select both the live and dead buttons in the adjust screen so that you are completely maximizing the size and brightness gates for both the live and dead cell population. This is mainly an issue if there are cells that appear in the image, but aren't being counted. It's best to expand the gates first, make sure all of the cells are counted, and then narrow them if you want to exclude cells of a certain size or certain brightness.

Answer Id:: E12364

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