Product FAQ

Can I purchase or use the QuantStudio™ 3 or QuantStudio™ 5 Real Time PCR System without a computer?

Answer

Yes, you can purchase the QuantStudio™ 3 or QuantStudio™ 5 Real Time PCR System without a computer. In this configuration, you can run the instrument through the touchscreen or the Design and Analysis application from Thermo Fisher Cloud.

Answer Id: E12055

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Product FAQ

When do I have to calibrate the QuantStudio™ 3 and QuantStudio™ 5 Real Time PCR Systems?

Answer

When your new QuantStudio™ 3 or QuantStudio™ 5 Real-Time PCR System arrives in your lab, it will be factory calibrated. The recommended calibration schedule is to recalibrate every 2 years.

Answer Id: E12056

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Product FAQ

Are there any additional components that need to be added to the media of the PSC Definitive Endoderm Induction Kit?

Answer

No other components are required.

Answer Id: E9380

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Product FAQ

How do I open the drawer in the QuantStudio™ 3 or QuantStudio™ 5 Real-Time PCR System?

Answer

The block drawer is opened and closed by using the ‘eject’ button on the touchscreen. Do not push or pull on the instrument drawer.

Answer Id: E12057

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Product FAQ

Can you provide some general tips to improve the CELLection™ release step?

Answer

Here are some suggestions:

- Make sure that pH of the RPMI + 1% FBS is not too high: DNase I works best between pH 7.0-7.4 (O2 exposure of RPMI will result in increased pH and the pH-indicator will turn blue).
- Do not use RPMI + 10% FBS during DNase I treatment. 10% FBS gives a lower cell yield than 1% FBS (might be caused by DNase I inhibitory factors in some batches of FBS).
- Make sure that the buffer contains Mg2+/Ca2+ required for DNase activity.
- DNase I must be treated gently. Vigorously stirring of the DNase solution can reduce the enzyme activity. Never vortex the DNAse solution.
- When selecting low numbers of cells, we recommend that you pre-coat tubes with RPMI + 10% FBS to reduce cell loss (cells are sticky and will easily attach to the tube wall during selection).
- DNase I has good activity at 20 degrees C, however, we recommend pre-warming the RPMI + 1% FBS to 37 degrees C before starting DNase I treatment because ‘room temperature’ varies from lab to lab.
- After cells are incubated with DNase Releasing Buffer it is essential that the bead-cell complexes are thoroughly pipetted before the magnetic separation to provide mechanical disruption to the DNA linker. Failure to pipette the cells will affect cell yield.

Answer Id: E12075

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Product FAQ

What are the critical parameters in my starting PSCs that influence the differentiation outcome?

Answer

It is critical that human PSCs are high quality, karyotypically normal, exhibit pluripotency markers, and kept under passage 100. Upon thaw, culture cells for at least one passage before seeding for definitive endoderm induction.

Answer Id: E9381

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Product FAQ

Can I use the Neon™ system for co-transfecting two or more plasmids?

Answer

We currently do not have data to support this, but co-transfection of different plasmids should work. However, the amount of DNA should be carefully titrated, since overloading the cells with plasmid DNA or using unfavorable ratios of the plasmids may cause toxicity. Therefore, we recommend starting optimizations of co-transfection experiments with low amounts of DNA followed by a stepwise increase. Various ratios of the plasmids should be tested if toxicity is observed.

Answer Id: E9118

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Product FAQ

With the QuantStudio™ 3 or QuantStudio™ 5 Real-Time PCR System, I want my file to go to my Thermo Fisher Cloud account once the run is complete for analysis. Is there a way for it to automatically upload?

Answer

Yes. To have the run file automatically upload to your Thermo Fisher Cloud account once complete, you will need to set the file destination on the touchscreen, prior to starting the run:
1. Before you start the run, select the ‘Properties’ tab from an open experiment
2. Touch ‘Edit’, Data Destination - Choose USB or Cloud
Alternatively, you could use Cloud Connect Utility to have all files from a specific hard drive folder location automatically upload to the cloud (for use with the QuantStudio™ Design and Analysis Desktop Software).

Answer Id: E12058

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Product FAQ

How can I remove guanidine hydrochloride using TCA, from my protein samples?

Answer

(1) Dilute 10 to 25 μL samples to 100 μL with H2O. Add 100 μL of 20% trichloroacetic acid (TCA) and mix. Store on ice for 20 min.
(2) Spin at 4 degrees C for 15 min in a microcentrifuge at maximum speed (15,000 x g).
(3) Carefully discard the supernatant and retain the pellet. Dry the tube by inverting it on tissue paper.
(4) Wash pellet with 100 μL ice-cold ethanol, dry, and resuspend in sample buffer. In case there are traces of guanidine HCl present, samples should be loaded immediately after boiling for 7 min at 95 degrees C.
Note: Presence of some TCA can give a yellow color as a consequence of the acidification of the sample buffer; titrate with 1N NaOH or 1M Tris-HCl pH 8.5 to obtain the normal blue sample buffer color.

To prepare 100% TCA: dissolve 1 kg of TCA in 454 mL water. Maintain in a dark bottle at 4 degrees C.

Answer Id: E4460

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Product FAQ

I ran my protein samples on a Precise™ gel and the protein bands were distorted. What could have gone wrong?

Answer

Here are possible causes and solutions:

1) Air bubbles in the sample wells, or between gel and cassette, or at the bottom of the cassette: Use a transfer pipette to displace the air bubbles from the sample wells.
2) Sample contains appreciable carbohydrate: Remove the carbohydrate by enzymatic or chemical means.
3) Sample contains lipoproteins: Use a gel with large pore size at top. Try addition of a non-ionic detergent.

Answer Id: E10614

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Product FAQ

Will these Quant-iT™ RNA kits work for siRNA or microRNA?

Answer

No, they are not accurate for such small fragments. The Quant-iT™ microRNA Assay Kit and Qubit™ microRNA Assay Kit are good tools for this application.

Answer Id: E8140

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Product FAQ

I am using counting beads to count cells, but I cannot find the beads on my scatter plots. What do I do?

Answer

The first thing to do is check your threshold and see if it is set on forward scatter. If so, the beads are probably being excluded by the threshold. Reducing the threshold setting should reveal your beads.

Answer Id: E14843

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Product FAQ

What is an FMO control in flow cytometry?

Answer

An FMO control (flow minus one) is a control in which you label cells or beads with every color in your panel, omitting one. Make one FMO control for each color in your panel. These controls are important for helping you properly set gates on your data.

Answer Id: E14817

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Product FAQ

Are there any stability problems arising from the fact that the whole Entranceposon is inserted into the target plasmid? Is the Entranceposon capable of further transposition inside host cells? How stable are the target plasmids that carry the Entranceposon?

Answer

The Entranceposonshave been designed so that the presence of the MuA Transposase enzyme is an absolute requirement for any transposition activity. The Entranceposons do not contain any genes from the bacteriophage Mu; only the DNA sequences from the right end of the Mu genome that are responsible for the transposase binding. However, the Entranceposons contain >50 bp inverted terminal repeats. To avoid instability resulting from homologous recombination between the repeats, the use of a recA mutant E. coli strain is recommended.

Answer Id: E8687

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Product FAQ

How was the substrate (Component A) in the EnzChek™ Lysozyme Assay Kit labeled?

Answer

The primary amines on the cell wall of Micrococcus lysodeikticus were conjugated to fluorescein.

Answer Id: E10786

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