How can I lyse my cells using a non-detergent based method or prepare tissue samples?

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Several methods, including mechanical disruption, liquid homogenization, sonication, freeze/thaw cycles, and manual grinding are commonly used to physically lyse cells.

Mechanical disruption: mechanical methods rely on the use of rotating blades to grind and disperse large amounts of complex tissue.
Liquid homogenization: cells are lysed by forcing the cell or tissue suspension through a narrow space, thereby shearing the cell membranes. The number of strokes and the speed at which the strokes are administered influences the effectiveness of homogenization methods.
Sonication: pulsed, high frequency sound waves agitate and lyse cells, bacteria, spores, and finely diced tissue.
The sound waves are delivered using an apparatus with a vibrating probe that is immersed in the liquid cell suspension. Mechanical energy from the probe initiates the formation of microscopic vapor bubbles that form momentarily and implode, causing shock waves to radiate through a sample.
Freeze/thaw: this technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer and then thawing the material at room temperature or 37 degrees C. This method of lysis causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing. Multiple cycles are necessary for efficient lysis. This method is commonly used to lyse bacterial and mammalian cells.
Mortar and pestle: manual grinding is the most common method used to disrupt plant cells. Tissue is frozen in liquid nitrogen and then crushed using a mortar and pestle.

Answer Id: E12795

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Why are protease/phosphatase inhibitors important during cell lysis?

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Cell lysis disturbs the carefully controlled cellular environment, allowing endogenous proteases and phosphatases to become unregulated. As a result extracted proteins become degraded or artifactually modified by the activities of these molecules.

Answer Id: E12796

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I want to perform a cell fusion assay, where one cell line is labeled with one color and the other cell line with another color, and combine with a nucleic acid stain. What do you recommend?

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A typical method is to label one cell line with orange fluorescent DiI C18 and the other cell line with green fluorescent DiO C18. These orange and green lipophilic cyanine dyes will stain the membranes of cells. Cells that fuse will then have both dyes, yielding a yellow color (when images are overlaid or cells are imaged in a dual-bandpass filter). These live cells can then be labeled with Hoechst 33342 (a cell-permeant blue DNA stain comparable in wavelength to DAPI), but only as an endpoint just before imaging (since DNA stains can interrupt DNA function).

Answer Id: E6447

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What does “Error - Cannot open the file because file contents are not recognized” mean?

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This issue can occur if duplicate samples are added to the experiment, if illegal characters are used in the experiment/file name, or if the file extension was changed to .eds from a different extension.
Please send the file to techsupport@thermofisher.com to attempt recovery.

Answer Id: E7214

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What is the first step in an experiment with the Gateway™ system?

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The first step is to create an Entry clone for your gene of interest. We have 3 options to do this: The first is by BP recombination reaction using the PCR Cloning System with Gateway™ Technology. This is recommended for cloning large (>5 kb) PCR products. We also have Gateway™ compatible TOPO™ Cloning vectors such as pCR™8/GW/TOPO™ and pENTR™/D-TOPO™. The final option is to use restriction enzymes to clone into a pENTR™ Dual Selection vector.

Answer Id: E3208

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Which protein assay is my lysis compatible with?

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Answer

Please check this Protein Assay Compatibility Table (https://tools.thermofisher.com/content/sfs/brochures/TR0068-Protein-assay-compatibility.pdf) to determine which protein assay would be most effective with your sample type.

Answer Id: E12797

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I am using ProLong™ antifade mounting medium. Do I need to let it cure before imaging? Do I need to seal the edges of the coverslip?

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You can image before it cures (hardens), and it will still slow photobleaching, but you have to let it cure overnight to get the best refractive index (resolution). There is no need to seal the edges. In fact, if you seal before it cures, it won't cure correctly. If you are archiving the slide for more than a month, though, seal the edges with resin, paraffin or VALAP (1:1:1 vaseline, lanolin, paraffin) after it cures or there may be slight discoloration along the edges over time.

Answer Id: E6448

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Can I use FFPE samples with the Ion AmpliSeq™ Exome RDY and Ion AmpliSeq™ Exome RDY S5 kits?

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Answer

The Ion AmpliSeq™ Exome RDY and Ion AmpliSeq™ Exome RDY S5 kits are not recommended for use with FFPE samples, as the amplicon target sizes (225-275 bp) are larger than we recommend for degraded DNA input. Increasing the input amount will not help, as the issue will be that some or many of the amplicon targets may not be amplifiable due to the degraded (fragmented) nature of the FFPE-derived DNA. This could result in amplicon drop out and incomplete coverage of the intended targets. Further, there could be issues with reproducibility across samples of differing levels of degradation. For example, some samples may produce sufficient results, while others may completely fail or produce sub-par results.

Answer Id: E11489

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What does “Error - Java.lang.IllegalArgumentException: One of the raw spectra is null” mean?

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This issue can occur if the “Start Run” button is clicked more than once or if illegal characters are used in the experiment/file name. You can recover the data yourself by overriding the calibration file with one that represents the calibration status of the instrument when you ran that particular file. Use the toolbar at the top to navigate: Analysis then Override Calibration then Use Calibration From Another File.

Answer Id: E7215

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From where does Gateway™ get its lambda nomenclature, and is it consistent with textbook nomenclature for lambda recombination?

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Answer

The Gateway™ nomenclature is consistent with lambda nomenclature, but we use numbers to differentiate between modified versions of the att sites (attB1, attB2, attP1, attP2, and so on). We have introduced mutations in the att sites to provide specificity and directionality to the recombination reaction. For example, attB1 will only recombine with attP1 and not with attP2.

Answer Id: E3209

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What HRM kit should I use for my application?

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Answer

We have two options for HRM reagents. The MeltDoctor™ HRM Master Mix contains all components (excluding template and primers) formulated for superior HRM performance across a wide range of genomic targets. It is recommended for most HRM application except methylation-sensitive HRM. The MeltDoctor™ HRM Reagent Kit provides individually packaged PCR components and the MeltDoctor™ HRM Dye required for high-resolution melt analysis that is recommended for methylation-sensitive HRM analysis (as it does not contain dUTP).

Answer Id: E7566

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I'm getting Mac-Microsoft_OLE2_ColsTrueOle1Class error. How do I fix this?

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Answer

To fix the problem, copy the attached “Microsoft™ libraries.sit” file to your desktop. Expand/un-compress the file with StuffIt Expander or other software. A folder called “microsoft libraries” is created, and in it are four files/extensions. Copy them into the Extensions folder in your System Folder (files called Microsoft Component Library, Microsoft OLE Automation, Microsoft OLE Library, Microsoft Structured Storage).

Answer Id: E4338

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I’m getting very few colonies after transformation of my TOPO™ cloning reaction. How can I increase the number of primary colonies?

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Answer

Please try the suggestions below to increase the number of colonies.
- Longer incubation of the TOPO™ cloning reaction at room temperature, provided that the 6X Salt solution is added to the reaction.
- Electroporation can give significant increases in colony numbers; often 10-20 fold higher. However, if doing electroporation, it is important that the TOPO™ reaction mix contains diluted Salt solution or, for best results, precipitated prior to transformation. For high primary transformants by electroporation it is recommended to:
- Add 100 μL double diH2O to the 6 μL TOPO™ reaction and incubate 10 more minutes at 37 degrees C.
- Precipitate by adding 10 μL 3 M Na-Acetate, 2 μL 20 μg/μL glycogen, 300 μL 100% ethanol. Place on dry ice or –80 degrees C for 20 min, spin at top speed in a microcentrifuge at 4 degrees C for 15 min. Wash pellet with 800 μL 80% ethanol, spin at top speed for 10 min, pour off ethanol, spin 1 min, and remove remaining ethanol without disturbing pellet. Dry pellet (air-dry or speed-vac).
- Resuspend pellet in 10 μL ddH2O and electroporate 3.3 μL of resuspended DNA according to a normal electroporation protocol. This electroporation protocol can yield up to 20 fold more colonies than chemical transformation of an equivalent TOPO-reaction. The addition of the 100 μL ddH2O followed by 10 mins incubation is not absolutely necessary, but it sufficiently dilutes the reaction and may help inactivate topoisomerase so that it is more easily electroporated.

Answer Id: E6761

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What lysis buffers do you offer for mammalian cells?

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Answer

Please see the various lysis buffers (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/cell-lysis-fractionation/cell-lysis-total-protein-extraction.html) we offer for total protein extraction from mammalian cells.

Answer Id: E12798

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I want to use MTT to determine cell proliferation. Do I really need a standard curve? Will the assay tell me percent of live versus dead cells?

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Answer

MTT is reduced only by live cells and the rate of dye reduction may vary from cell to cell based on their life cycle stage, age, health and other factors. This assay would not reveal the number of dead cells or a ratio of live to dead cells. A standard curve is recommended to determine an approximate number of live cells per sample.

To obtain a ratio of live to dead cells, use a two color fluorescent assay, such as LIVE/DEAD™ Viability/Cytotoxicity Kit (Cat. No. L3224) or differences in intensity of a single fluorescent color, using a flow cytometer and the LIVE/DEAD™ Fixable Kits.

Answer Id: E6449

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