Product FAQ

When editing a cycle on the Procise™ System, I can readily delete a step, but cannot seem to insert a function into the cycle. What am I doing wrong?

Answer

When you edit a flask cycle, be sure to highlight a flask function in order to insert it. Similarly, when editing a cartridge cycle, highlight a cartridge function in order to insert it.

Answer Id: E1257

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

My neural stem cells (NSCs) died after seeding. What went wrong?

Answer

Here are some reasons why your cell culture could have failed:

- Did not store cells correctly: These cells should be stored in liquid nitrogen.

- Medium used was not the recoomended one: The complete medium of Gibco™ H9-derived NSCs (Cat. No. N7800) and Gibco™ StemPro™ NSCs (Cat. No. A15654 or A15655) have different formulations. Extra components (heparin and ascorbic acid) need to be added into the medium to culture StemPro™ NSCs.

- Did not thaw cells correctly: Do not thaw the cells for longer than 2 mins at 37 degrees C. After cells have thawed, transfer to 50-mL tube first, then add pre-warmed complete medium drop-wise (approximately 1 drop per second) while swirling the tube.

- Did not count cell viability after thawing cells and did not seed them appropriately: You need to count cell viability with trypan blue. At least 1 x 10E6 viable cells/mL are provided in each vial. For H9-derived NSCs, recommended seeding density is > 1 x 10E5 viable cells/cm2. For StemPro™ NSCs, recommended seeding density (suspension) is >7 x 10E4 viable cells/mL.

- Plate is not coated or has been coated incorrectly: For H9-derived NSCs and adherent culture, you need to properly coat the tissue culture plate with Gibco™ Geltrex™ Matrix, fibronectin, or poly-L-ornithine/laminin, following instructions for coating. For StemPro™ NSCs, we recommend that you grow them in suspension culture, as adherent culture would trigger differentiation.

Answer Id: E12580

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Why were our NSCs stained with neuron-specific markers such as GFAP or Beta III-tubulin?

Answer

NSCs do not stain with neuron-specific markers. If staining was observed, this could have resulted from antibody non-specific staining. We recommend checking antibody specificity and the staining protocol:

- Titrate primary antibody to find the optimal antibody working concentration.
- Change the permeabilization protocol, because a high concentration of permeabilization reagent(s) could result in high background.
- Use enough blocking solution such as 5-10% serum to block non-specific staining.
- Include isotype control.

Answer Id: E12581

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Which panel can the TaqMan™ OpenArray™ Real-Time PCR Master Mix be used with?

Answer

The Master Mix has been optimized for use with the TaqMan™ OpenArray™ gene expression panels, and yields reproducible, specific and sensitive results.

Answer Id: E10077

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Why do you recommend a non-frost-free freezer for antibody storage?

Answer

Non-frost-free freezers are recommended for antibody storage because they do not go through automatic defrost cycles. To keep a freezer compartment frost-free, the freezer actually defrosts itself periodically with heating coils. Temperatures inside the freezer can rise to 0 degrees C and above during the defrost cycle. If antibodies are subjected to these temperatures for a long enough time, they can undergo a partial thaw. This happens especially at the interface between the air in the sample container and the surface of the frozen contents. Since air warms faster than water, proteins at the interface are affected more than those in the depths of the tubes.

Even if you are using a non-frost-free freezer, you should pay attention to where your antibodies are stored inside it. It is best to store them in an area of the freezer compartment that experiences the least temperature fluctuation, which is usually in the center in the back of the freezer. Do not store antibodies near the front of the freezer compartment or on a shelf in the freezer door, where they are exposed to room temperature every time the freezer door is opened. Make sure as well that the antibody container is tightly sealed. This can help to prevent water loss from the antibody via sublimation (conversion of ice directly to water vapor that escapes from the containers). This slow process is basically freeze-drying at normal atmospheric pressure, and it can lead to damage to the antibody.

Answer Id: E12599

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Why are there no viable primary neuron cells/astrocytes/rat glial precursor cells after thawing the cell stock?

Answer

These cells are very fragile. We recommend following the procedure in the manual and using the correct medium. Fast thawing is the key for healthy culture. There are several critical points to consider:

- Pre-rinse all materials with medium before use. Do not use PBS, DPBS, or HBSS to rinse because they do not contain proteins.
- Thaw cells quickly and do not expose cells to air.
- Transfer cells to a pre-rinsed tube first, then slowly add medium in a drop-wise manner. Do not add the full amount of medium to the cells at once because this may result in osmotic shock and decreased cell viability.
- Use pre-warmed complete growth medium and correct seeding density.
- Matrix coating is required for some cell cultures.
- For primary neuron cells, do not centrifuge the cells as they are extremely fragile upon recovery from cryopreservation.

Answer Id: E12582

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

What is the difference between TRIzol™ reagent and TRIzol™ Plus reagent?

Answer

TRIzol™ Plus reagent combines TRIzol™ reagent and a column-based purification system in order to isolate the highest quality RNA. The column purification helps to remove 18s RNA and tRNA, removes any trace of phenol, and provides for double gDNA removal.

Answer Id: E6548

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

How does SYPRO™ Ruby Protein Gel Stain compare to other total protein stains?

Answer

Since SYPRO™ Ruby Protein Gel Stain binds primarily to basic protein residues instead of SDS, it can be used as an endpoint stain (direct, saturation binding with a low off-rate) whose performance is not affected by deviations in fixation, staining or destaining solution volumes or incubation times, but just the amount of protein that is present. The strong 50% methanol/7% acetic acid fixation in the SYPRO™ Ruby stain protocol removes most SDS bound to proteins and in the gel matrix, so that the signal is due to direct protein binding. Stains that bind protein indirectly via SDS/protein association (such as Deep Purple, Lucy, SYPRO™ Orange/Red/Tangerine, Nile Red dyes) show higher protein to protein and gel to gel variation depending on how much SDS is bound to the protein and present in the gel matrix. Proteins that do not bind SDS to saturation, such as heavily glycosylated or phosphorylated proteins will show reduced staining with indirect SDS binding protein stains.

Answer Id: E11128

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

I left my DNA-ExpiFectamine™ 293 transfection complexes sit longer than 20 minutes. Will they be okay?

Answer

The best transfection efficiencies are obtained when transfection complexes are used fresh. However, they should be stable for at least an hour.

Answer Id: E9264

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

How should I adjust the Platinum™ Pfx DNA Polymerase protocol if I am trying to generate an amplicon greater than 2 kb, or if I am starting with long PCR primers?

Answer

To generate amplicons greater than 2 kb, use 2.5 units of Platinum™ Pfx polymerase (instead of 1 unit), decrease the extension temperature to 68 degrees C, and increase the extension time to 1 kb/minute.

If long PCR primers are used with Platinum™ Pfx polymerase, increase the magnesium concentration in the reaction to 1.5 mM.

Answer Id: E3053

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

How does the SYBR™ GreenER™ Dye Chemistry work?

Answer

The SYBR™ GreenER™ dye binds to double stranded DNA formed during PCR. During PCR, the polymerase amplifies the target sequence which creates the amplicon. As PCR continues, the dye binds to each new copy of double-stranded DNA, leading to more amplicons being created. The result is an increase in fluorescence intensity proportional to the amount of double-stranded PCR product produced.

Answer Id: E10078

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Will the formulations containing GlutaMAX™ supplement change with respect to L-glutamine content?

Answer

In all media containing GlutaMAX™ supplement dipeptides as a substitute for L-glutamine, concentration is equimolar with the L-glutamine in the original formulation.

Answer Id: E2984

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Do I need to test my siRNA in vitro before using it in vivo?

Answer

Yes! We recommend that you test at least 3 different siRNA duplexes in vitro at several different concentrations and use the siRNA duplex with the highest potency for your in vivo experiments. When moving from in vitro to in vivo experiments, it is also important to double check the species specificity of the duplex.

Answer Id: E9805

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

My gel stained with Pro-Q™ Emerald 300/488 Glycoprotein Gel Stain is showing poor staining of my glycoproteins and the glycosylated CandyCane™ molecular weight standard. What is causing this and how can I improve the signal?

Answer

- Poor staining can be caused by the presence of primary amines, such as from Tris or glycine, that will also react with the aldehyde/ketone groups generated by periodate oxidation. This effectively caps the reactive groups, leaving no reactive sites for Pro-Q™ Emerald dye to bind. To remove any amine contamination, increase the volume or number of incubations in fresh fixative and then increase the volume or number of washes in wash buffer.
- Poor staining can also be due to inadequate removal of the periodic acid oxidation solution. Increase the volume or number of washes after the oxidation step to ensure adequate removal of periodic acid.
- Poor staining can also be due to inadequate oxidation of glycoprotein sugars. Increase the volume of periodic acid oxidation solution.

Note: Do not increase the number or incubation time of the periodic acid oxidation in excess of 30 minutes for small-format gels. Excessive periodic acid oxidation could result in increased staining of non-glycosylated proteins. In general, large format, unusually thick or very high percent acrylamide gels may require additional incubations or wash steps for optimal signal and sensitivity of staining with Pro-Q™ Emerald Glycoprotein Gel Stain.

Answer Id: E11299

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

Is my antibody stable at room temperature?

Answer

Generally, antibodies can be left at room temperature for up to a week without loss of activity. Hence, we ship many of our antibodies at ambient temperature. However, longer storage at room temperature or higher is not recommended, and we can’t guarantee the performance of the antibody under such circumstances. Please contact Technical Support at techsupport@thermofisher.com if you have further questions about antibody stability.

Answer Id: E12600

Was this answer helpful?

Yes
No
Thank you for your response