Manual / Product Insert

LIVE/DEAD BacLight Bacterial Viability Kits

Version: 07-15-2004

Manual / Product Insert

Quick Reference: ProteinSEQ Protein A Quantification Kit; Workflow for Standard PCR plates

Version: MAN0013526 Rev.A (06Dec2015)
Catalog #

Manual / Product Insert

MSQ Plus Mass Detector Calmix Kit Preparation Guide Revision A

Version: FEB.2016
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Citations & References

Nanoengineered structures for holding and manipulating liposomes and cells.

  • Authors: Wilson CF, Simpson GJ, Chiu DT, Strömberg A, Orwar O, Rodriguez N, Zare RN
  • Journal: Anal Chem (2001) 73:787-791
  • PubMed ID: 11248893
Catalog #
  • C2213(Discontinued)

Manual / Product Insert

Quick Reference: ProteinSEQ Protein A Quantification Kit; Workflow for FAST PCR plates

Version: MAN0013525 Rev.A (06Dec2015)
Catalog #

Manual / Product Insert

QuantiGene miRNA Singleplex Assay User Manual

Version: JAN 2017

Product Literature

Specification Sheet: Is there an easier and more efficient way to transfer liquids between various labware formats, than using traditional handheld pipettes?

Product FAQ

What is your recommended protocol for generating ss phagemid DNA?

Answer

The following protocol can be used to prepare ssDNA from DH12S or DH5aF', DH5aF'IQ, DH11S cells (this strain not currently offered for sale). Use a stock of the helper phage M13KO7 (1) that is of known titer. For convenience, DH12S cells are supplied with M13KO7 helper phage. DH12S cells are both endA positive, so single stranded DNA isolated from these strains tend to be less contaminated with double stranded rf DNA.

Small-Scale Preparation of ss Phagemid DNA:
1. Pick a single colony of cells containing phagemid DNA and resuspend in 2 mL TBG (1.2% tryptone, 2.4% yeast extract 0.4% glycerol, 17 mM KH2PO4 and 55 mM KH2PO4 and 20 mM glucose) containing 100 µg/mL ampicillin in a 15 mL tube.
2. Immediately add 10 µL M13KO7 helper phage stock at 10E11 pfu/mL.
3. Incubate cells at 37 degrees C with vigorous agitation (275 rpm) for 2 hrs.
4. Add kanamycin to a final concentration of 75 µg/mL and incubate cells at 37 degrees C with vigorous agitation (275 rpm) for 18 to 24 hrs.
5. Transfer 1.5 mL of culture to a sterile microcentrifuge tube and pellet cells by centrifuging at 14,000 x g for 10 min at 4 degrees C.
6. Transfer supernatant to fresh microcentrifuge tube and repeat the centrifugation.
7. Transfer 1.2 mL supernatant to a fresh microcentrifuge tube and add 300 µL of 2.5 M NaCl in 40% PEG 4000.
8. Vortex and incubate on ice for 15 min.
9. Centrifuge at 14,000 x g for 15 min at 4 degrees C.
10. Resuspend the pellet in 50 µL TE and phenol extract to remove the viral coat.
11. Use 10 µL of the final 50 µL volume for gel analysis.
This protocol yields 0.5 to 1 µg ss phagemid DNA.

Large-Scale Preparation of ss Phagemid DNA:
1. Resuspend a single colony in 5 mL of TB or LB broth containing 100 µg/mL ampicillin in a 15-mL tube.
2. Shake at 37 degrees C and 275 rpm overnight.
3. Add 100 µL of the overnight culture to 200 mL LB broth and 100 µg/mL ampicillin in a 1 L flask. Incubate at 37 degrees C with shaking (275 rpm) for 3 hrs.
4. Add 200 µL of M13KO7 helper phage (1 x 10E11 pfu/mL) to the culture and continue to incubate for 2 hrs.
5. Add 1.5 mL of 1% (w/v) kanamycin to the cells for a final concentration of 75 µg/mL. Incubate the infected cells for an additional 18 to 24 h at 37 degrees C.
6. Centrifuge this culture at 16,000 x g for 15 min at 4 degrees C .
7. Filter the supernatant through a 0.2 µm sterile filter into an autoclaved centrifuge bottle. Add 40 µL of DNase I (50 units/µL) and incubate at room temperature for 3 hrs. This step should remove any residual ds plasmid DNA contamination.
8. Transfer 100 mL of the supernatant to another centrifuge bottle. Add 25 mL of 2.5 M NaCl in 40% PEG 4000 to each of the centrifuge bottles containing the supernatant.
9. Vortex the mixture, incubate on ice for 1 h, and centrifuge at 16,000 x g for 20 min at 4 degrees C.
10. Carefully discard the supernatant. To fully drain off the remaining solution from the pellets, place the bottles on an angle, with the pellet side facing up for 10 to 15 min. Remove the solution with a sterile Pasteur pipet.
11. Resuspend the pellets in 2 mL of TE buffer. Add 10 µL of proteinase K solution (20 mg/mL), 20 µL of 10% SDS, and incubate this mixture at 45 degrees C for 1 hr.
12. Transfer the digested mixture to three microcentrifuge tubes and extract four times with an equal volume of phenol:chloroform: isoamyl alcohol (25:24:1), precipitate with ethanol, and dissolve in 100 µL TE buffer.
13. Freeze the solubilized DNA at -20 degrees C for 1 hr and centrifuge in a microcentrifuge at 14,000 x g for 15 min at 4 degrees C.
14. Transfer the supernatant containing the ss plasmid DNA to a fresh tube and discard the polysaccharide pellet. Store the ssDNA at 4 degrees C.
15. Determine the DNA concentration (OD260).
This protocol yields ~100 to 200 µg ss phagemid DNA.

(1) Vieira, J. and Messing, J. (1987) Methods in Enzymology 153, 3.

Answer Id: E4161

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Product FAQ

What are small particles that appear after a few days when growing Sf9 cells? I am not using Gibco Fungizone reagent, but I am using gentamicin. Are they yeast?

Answer

Yellow particles could be cell organelles, aggregates, or debris. We see this when we first thaw frozen cells. To avoid this, you can let the shaker culture sit for 5 minutes, then transfer top 1/3 to a new flask, making sure to count cells first. You can also use heparin at up to 200 U/mL to decrease aggregation. Pluronic solution at a final concentration of 0.2% can also be used to decrease shearing, and increase shake speed to 100-120 rpm. Recently thawed cells seem to be breaking up and releasing small vesicles, as observed under high magnification. To reduce the amount of those small particles, cells need to be rapidly but completely defrosted for successful thawing to take place. Also:

1. Place vial on ice during transfer from water bath to sterile hood.
2. Pipette as gently as possible because cells shear easily due to larger surface area.
3. Cells may not have been placed in cold media after removal from defrosted vial into flask.
4. Media may not have been changed after 30-45 minutes once a majority of cells had attached. Media change should be with pre-warmed media (27degrees C). 10% DMSO in freezing medium will kill the cells if left on them for long periods of time (1 hour seems to be a maximum).
5. Lastly, cells should be checked for contamination. To do so, plate a small portion of culture in a T-25 flask and incubate for 3 days, checking for cloudiness.

Answer Id: E11957

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Product FAQ

With the Thermo Scientific Power Blotter, I got Inconsistent transfer of my proteins. Why is this?

Answer

Here are possible causes and solutions:

- Membrane or filter paper was insufficiently equilibrated in Thermo Scientific 1-Step Transfer Buffer: Equilibrate membrane and filter paper in Thermo Scientific 1-Step Transfer Buffer before transfer for a minimum of 5 minutes. Use a sufficient amount of buffer for the equilibration step.
- Insufficient transfer time: Increase transfer time from 7-10 minutes to 10-12 minutes.
- PVDF membrane was not pre-wetted with methanol or ethanol: Wet PVDF membrane with methanol or ethanol and equilibrate for 10-15 minutes in Thermo Scientific 1-Step Transfer Buffer before transfer.
- Air bubbles trapped between gel and membrane: When assembling transfer stack, use a roller or pipette to remove any air bubbles between the gel and the membrane.
- Filter paper used in the fast transfer exceeded 1.8 mm: Use filter paper <1.8 mm thick.

Answer Id: E11647

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Product FAQ

I performed a western transfer and see the appearance of diffuse bands and swirling patterns on the membrane. What could have happened?

Answer

The swirling and diffuse banding patterns are typical of molecules moving laterally before binding to the membrane during transfer. Here are possible causes and solutions:

- Poor contact between the gel and the membrane: The gel should be attached to the membrane through capillary action. To ensure that this happens, make sure that you roll over the surface of each layer of the gel/membrane sandwich with a glass pipette to ensure good contact between the gel and the membrane. It is helpful to use a disposable pipette to place some extra transfer buffer on the surface of each layer as the sandwich is being made. Also, the pads need to be fully saturated (push down with gloved hand when they are placed in transfer buffer to make sure there are no air bubbles.)
- Under-compression of the gel: The gel/membrane assembly should be held securely between the two halves of the blot module. Try adding another pad or replace any pads that have lost their resiliency with fresh ones.
- Over-compression of the gel: A good indication of over-compression is if the gel has been excessively flattened. In the event that the sandwich is over-compressed, remove enough pads so that the blotter can be closed without exerting excess pressure on the gel and membrane.
Note: The height of the uncompressed pads should be 0.5-1.0 cm above the level of the sealing gasket.

Answer Id: E11604

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Product FAQ

Should I use top agar or agarose to plaque my phage stocks before PCR screening?

Answer

Agar contains sulfated polysaccharides that can inhibit Taq DNA polymerase. Use agarose in the lambda top mixture and limit the agarose carry over when plaques are picked. Isolate the desired plaque using the following procedure:

(1) Punch through the agarose top layer with a sterile pasteur pipette.

(2) Transfer the plaque to a tube containing 12 µL SCL solution [10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 50 µg/mL proteinase K] and incubate for 15 min at 55 degrees C.

(3) Inactivate proteinase K for 15 min at 80 degrees C.

(4) Add 20 µL of deionized water and centrifuge at 12,000 x g for 3 min.

(5) Transfer the supernatant to a new tube.

Answer Id: E2971

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Product FAQ

When should I use the Versette Automated Liquid Handler vs. the Multidrop Combi Reagent Dispenser?

Answer

The Versette Automated Liquid Handler is the perfect solution for applications that require aspiration of liquid, plate transfers/reformatting, mixing, serial dilutions, and other common plate preparations performed using hand held pipettes in the lab.

The Multidrop Combi Reagent Dispenser should be used for fast bulk reagent dispensing and plate preparation.

Answer Id: E17161

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Product FAQ

What causes empty spots on my membrane after transfer?

Answer

Here are possible causes and solutions:

- Presence of air bubbles between the gel and the membrane preventing the transfer of proteins. Be sure to remove all air bubbles between the gel and membrane by rolling a glass pipette over the membrane surface.
- Expired or creased membranes used. Use fresh, undamaged membranes.

Answer Id: E11605

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Product FAQ

How do I remove the beads after the T cells are activated?

Answer

After 3-4 days in culture, most of the Dynabeads magnetic beads have detached from the cells.

For expansion protocols, we recommend that you remove the beads with the help of a magnet between days 8-12 depending on the assay. Pipette the bead:cell complexes thoroughly before applying to the magnet for 2 minutes, and transfer the supernatant with the released cells to a new tube. Discard the used beads and add fresh beads for re-stimulation.

For short-term activation, the cells can be lysed while they are still on the beads for molecular assays. Between days 1-3, the beads form strong clusters with the cells, thus it is hard to remove the beads in this period without losing a lot of the cells.

Answer Id: E12151

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Product FAQ

How do I process serum samples for use on the Luminex assay platform?

Answer

Serum samples should be collected in pyrogen/endotoxin-free tubes. Whole blood should be allowed to sit at room temperature for 15-30 min to clot. Spin at 1,000-2,000 x g for 10 min in a 4 degrees C refrigerated centrifuge to separate the cells. Transfer the supernatant to a chilled clean polypropylene tube with a sterile Pasteur pipette. Maintain the samples at 2-8 degrees C while handling.

If the serum is to be analyzed at a later date, apportion it into 0.5 mL aliquots and store at -80 degrees C. Avoid multiple freeze-thaw cycles. When possible, avoid the use of hemolyzed or lipemic sera. We recommend that upon thawing the samples be clarified by centrifugation (14,000 rpm for 10 min) and/or filtered, prior to analysis, to prevent clogging of the filter plates and/or probe. Follow the assay procedure provided with the kit for appropriate dilutions.

Answer Id: E12640

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Product FAQ

How can I extract peptides from NuPAGE gels for MALDI/MS analysis?

Answer

To extract peptides from NuPAGE gels perform the following protocol:

1) Dehydrate the gel band in 100% methanol for 5 min at room temperature.

2) Rehydrate the gel band in 30% methanol for 5 min.

3) Wash the gel band twice in ultrapure water for 10 min.

4) Wash the gel band three times with 100 mM ammonium bicarbonate containing 30% acetonitrile for 10 min.

5) After the last wash, cut the gel into small pieces and wash the gel pieces in ultrapure water.

6) Dry the gel pieces in a SpeedVac concentrator for 30 min

7) Resuspend the gel pieces in 50 mM ammonium bicarbonate. Add approximately 5 µL buffer per square millimeter of gel. There should be sufficient buffer to cover the gel pieces.

8) Add 5-10 ng/µL trypsin and incubate overnight at 37°C.

9) Centrifuge at maximum speed in a microcentrifuge for 1 min and transfer supernatant to sterile microcentrifuge tube using a clean pipette tip.

10) Extract remaining peptides from the gel with 10-20 µL 50 acetonitrile containing 0.1 trifluoroacetic acid at room temperature. Combine this extract with the supernatant from step 9.

11) Concentrate the sample from step 10 to 4-5 µL using a SpeedVac concentrator and proceed to MALDI/MS analysis. Be sure to include a control sample for MALDI/MS analysis.

Answer Id: E4145

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Product FAQ

Are there any general recommendations or points to consider when setting up a Luminex assay?

Answer

TIP #1: Use a bunch of paper towels to blot the plates after each wash step. After removing the plate from the vacuum manifold, there are usually droplets of liquid still present on the bottom of the plate. By blotting you remove this, eliminating the possibility that the droplet will act as a wick when in contact with another surface and leak volume out of wells.

TIP #2: Shake the plates as fast as possible to allow for maximum vortex without spillage. The beads are conjugated with monoclonal antibodies over their entire surface area. Think of each bead as being the same as one well in an ELISA plate. Since the beads are heavy and sticky, they sink to the bottom of the well if left in standing solution and then clump together. Once they are clumped together on the plate, you cannot separate them and you will have aggregation problems during the plate read. To avoid this, after sonicating them, we shake the beads on the plate at a speed that allows for maximum vortex without spillage.

TIP #3: As a rule, the strength of the vacuum filtration setup should be such that it takes three seconds to empty each well of wash solution. Since there is extraordinary variation in the strength of vacuum lines and pumps, it is safer for you to use this guideline rather than a value. Since the filter membrane is relatively fragile, applying too much pressure, even for an instant can cause tears and subsequent bead loss (vacuum flow should be established before placing the plate on the manifold) and DO NOT EXCEED 5 mm of Hg . The three-second-count rule is an easy and safe guideline.

TIP #4: Spin all samples down prior to loading. Another feature about filter plates is the possibility for them to become clogged. This happens when the sample has visible precipitate or aggregated matter, which most commonly occurs for serum and plasma samples (based on collection techniques). While we typically do not recommend spinning down TCS samples, we have seen this sample type clog a plate as well. Since clogs cannot be resolved once they occur, meaning a loss of data for the well, it is better to prevent the clog by spinning the samples down prior to loading. Generally, a 1 min high-speed spin is sufficient for most samples.

TIP #5: Preload samples onto a nonbinding/low binding microtiter plate to allow for easy transfer and quick loading in the assay. A 96-well plate represents a large number of samples, particularly when you are not running duplicates. Because binding begins immediately once a sample is added to a well, the difference in incubation time between the first and last well can be substantial. This can have an effect on the sample values, along with exposing the beads to excessive amounts of light during loading. The easiest way to avoid this, is to preload an aliquot of samples onto a microtiter plate and then simply transfer them with a multi-channel pipettor at the appropriate point in the assay.

TIP #6: If a well is clogged, use the bottom of a 15 mL conical tube to very gently press the bottom tip of the well from left to right once before attempting to apply vacuum again. A clogged well only occurs following the sample incubation step. When this happens, it is important that you do not raise the vacuum pressure, since this will risk tearing the other wells. Instead, use the tip of a 15 mL conical tube to gently press the bottom tip of the well funnel from left to right once. Try to aspirate the sample again, and if that does not work repeat the gentle pressing. After two or three attempts, you can consider the well permanently clogged and the data lost. It is not possible to dissolve major clogs like this with high detergent, because the detergent will also disrupt the antibody binding. It is also not possible to manually empty and wash the well from the top end, since beads will be removed each time until the point where there are none left to be analyzed.

Answer Id: E5139

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Product FAQ

What is the smallest sample volume I can use when extracting RNA with TRIzol Reagent?

Answer

Small volumes (0.5-0.8 mL) of TRIzol Reagent have been used successfully for 10^2 to 10^5 cells, but if small volumes are used, we recommend using smaller tubes in order to have the tallest possible column of aqueous phase. The taller the column of liquid, the less likely that contamination from the interphase will occur.

Here is a protocol for isolation of RNA from small quantities of tissue (1-10 mg) or cells (100-10,000):
1. Add 800 µL TRIzol Reagent to the sample. Homogenize cells by pipetting repeatedly. Add 200 µg glycogen (Cat. No. 10814010) directly to the TRIzol Reagent. If processing tissue, pulverize in liquid nitrogen first and then add 800 µL TRIzol Reagent containing 200 µg glycogen (final concentration 250 µg/mL) followed by vigorous vortexing or power homogenization.
2. Place at room temperature, cap the vial, and vortex at high speed for 10 seconds. Make sure the TRIzol Reagent wets the side of the vial in order to solubilize any sample that may be remaining on the walls.
3. Shear the genomic DNA in the sample by passing twice through a 26-gauge needle connected to a 1 mL syringe. Using the syringe, transfer the sample to a sterile 1.5 mL microcentrifuge tube.
4. Add 160 µL of chloroform (or 49:1 chloroform:isoamyl alcohol) to each sample and vortex up to 30 seconds. Centrifuge at maximum speed in a microcentrifuge for 5 minutes to separate the phases.
5. Transfer the upper aqueous phase to a fresh tube and add 400 µL ice-cold isopropanol. Allow the samples to precipitate at -20 degrees C for 1 hour to overnight. Pellet the RNA by centrifugation at maximum speed in the microfuge for 15 minutes at room temperature.
6. Decant the supernatant. Wash the pellet in 200 µL of 70% ethanol and centrifuge again for 10 minutes at maximum speed. Decant the supernatant, removing as much as possible without disturbing the pellet. Dry the RNA pellet.
7. Resolubilize the pellet in 30-50 µL RNAse-free deionized water. If tissue is high in RNAses (e.g., adrenal gland, pancreas), resuspend in 100% deionized formamide. Be sure to vortex or pipette the sample up and down to ensure that the pellet is fully resolubilized. Store at -70 degrees C.

Answer Id: E7833

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Product FAQ

I performed an ELISA assay, and the A450 readings for my duplicate wells were very inconsistent. What could have gone wrong?

Answer

Here are possible causes and solutions:

Errors in pipetting the standards or samples or in subsequent steps. Always dispense into wells quickly and in the same order. Do not touch the pipette tip on the individual microwells when dispensing. Use calibrated pipettes and the appropriate tips for that device. Check for any leaks in the pipette tip.
Repetitive use of tips for several samples or different reagents. Use fresh tips for each sample or reagent transfer.
Wells have been scratched with the pipette tip or washing tips. Use caution when dispensing into and aspirating out of microwells.
Liquid transferred from well to well during incubations. Adjust the orbital shaker or check for correct rotator rpm. Peel the adhesive plate cover off carefully.
Incorrect volumes of materials dispensed into the microwells. Follow the protocol for dispensing volumes of reagents. Check calibration of the pipettes.
Standard diluted with the serum, culture medium, or other buffer. Dilute the standard with the standard diluent buffer provided in the kit.
Particulates or precipitates present in the samples. Remove any particulates/precipitates by centrifugation prior to dispensing into the assay.
Dirty microwells: visible debris within or on bottom of microwells. Inspect the microwells and invert the plate to remove debris. Wipe the bottom of the plate with an absorbent tissue after each wash step. Never insert tissue into the microwells.
“Edge effect” due to uneven temperature between the outer-edge wells and the wells in the center of the plate. Seal the plate completely with a cover during incubations, and place the plate in the center of the incubator when 37 degrees C incubation is indicated.

Answer Id: E12634

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Product FAQ

How do I process plasma samples for use on the Luminex assay platform?

Answer

Separate the cells from the plasma samples by centrifugation at 2,000 x g for 10 min in a refrigerated centrifuge. Centrifugation at this force is necessary to deplete the sample of platelets. Transfer the supernatant to a chilled clean polypropylene tube with a sterile Pasteur pipette. Maintain the samples at 2-8 degrees C while handling.

If the plasma is to be analyzed at a later date, apportion it into aliquots in polypropylene microcentrifuge tubes and store at -80 degrees C. Avoid multiple freeze-thaw cycles. When you are ready to analyze them, allow the samples to thaw on ice. All plasma samples should be clarified by centrifugation (14,000 rpm for 10 min at 4 degrees C) in a refrigerated microcentrifuge immediately prior to analysis. Follow the assay procedure provided with the kit for appropriate dilutions.

Answer Id: E12641

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Product FAQ

How do you prepare blood cells for chromosome analysis?

Answer

Phytohemagglutinin Assay:

Lymphocytes are differentiated cells which normally do not undergo subsequent cell divisions. By culturing lymphocytes in the presence of a mitogen (KaryoMAX Phytohemagglutinin (M-Form) (PHA), Cat. No. 10576), they are stimulated to replicate their DNA and enter into mitosis. After an optimum time of the cells being cultured (46 h for a newborn and 68 h for an adult), a mitotic inhibitor, KaryoMAX COLCEMID Solution (Cat. No. 15210 or 15212), is added to the lymphocyte culture for 20 min. The addition of COLCEMID to dividing cells acts to prevent the synthesis of spindle fibers and, therefore, to stop mitosis in metaphase. Metaphase is the optimum phase of mitosis for microscopically visualizing the chromosomes. By submitting cells to a hypotonic solution and a series of fixation steps, metaphase chromosomes can be microscopically observed and analyzed.

As a quality control measure, each lot of Phytohemagglutinin is tested as a chromosome reagent for the examination of metaphase spreads used for cytogenetic studies. This reagent is evaluated by supplementation to an approved, non-phytohemagglutinin containing chromosome medium. These samples are then supplemented with freshly collected human peripheral blood and have been found to be acceptable in their ability to produce blastogenesis with human lymphocytes when compared to a previously tested control.

Test Procedure:

1. The required volume of peripheral blood is collected aseptically in a sodium heparinized vacutainer tube or syringe.
2. Add 10 ml of either PB-MAX Karyotyping Medium (Cat. No. 10386) to each sterile T-25 flask to be set up for the assay.
3. Add 0.75ml blood to each tube.
4. Incubate flask in CO2 incubator for 48 to 68 h with caps loose.
5. Add 0.05-0.1 ug/ml COLCEMID to each flask for a 15 minute incubation.
6. After 15 min, transfer flask contents to a 15 ml centrifuge tube and spin down at 1,200 rpm for 5 min.
7. Remove supernatant and resuspend pellet.
8. Add 10 ml of 0.068 M KCl to pellet and gently mix. Allow to sit at room temperature for 15 min. Add 0.5 ml of fixative (three parts absolute methanol to one part glacial acetic acid). Gently mix with pipette.
9. Centrifuge for 5 min at 1,200 rpm. Aspirate off supernatant. Add 10 ml of fixative, mix, and let sit at room temperature for 10 min.
10. Repeat centrifugation step. Add 5 ml of fixative, mix, and let sit for 10 min at room temperature.
11. Centrifuge and aspirate off supernatant. Add 5 ml of fixative and incubate for 10 min at 4°C.
12. Centrifuge at 1,200 rpm for 7 min. Aspirate off supernatant. Gently resuspend in fixative.
13. Prepare slide by placing 6 to 8 drops of cell suspension on slide. Use warming plate to dry slides.
14. Once all traces of moisture have disappeared, you can begin staining procedure:
- Stain with Giemsa Stain for 6 to 8 min, or any other appropriate stain.
- Remove slides from stain and rinse under running distilled water.
- Allow slides to thoroughly dry and examine slides for well-spread metaphases.

Record number of cells and at the same time record the number of these cells which are in metaphase. At least 500 cells must be counted for a valid assay. Statistically determine the Mean Mitotic Index (a quantitative measure) and Mean Banding Resolution (a qualitative measure). The test sample values must favorably compare to the reference control values.

Answer Id: E4306

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Product FAQ

How can I use Dynabeads MyOne SILANE magnetic beads for isolation of total RNA?

Answer

Please see the example protocol for isolation of total RNA from cultured cells (with enzyme):

Add 20 µl (20 mg/ml) Proteinase K prepared in Elution Buffer to 1 million freshly harvested cultured cells delivered in a volume of 50 µl. Mix and incubate at room temperature for 2 minutes.
Add 350 µl of Lysis/Binding Buffer and mix by pipetting. Incubate at room temperature for 10 minutes.
Note: Do not add Proteinase K directly to Lysis/Binding Buffer without cells .
Note: The vial containing Dynabeads MyOne SILANE magnetic beads should be resuspended (e.g. vortex) to a homogenous suspension prior to use. Leave on a roller until use.
Add 50 µl Dynabeads MyOne SILANE suspension (40 mg/ml) to the mixture.
Add 400 µl of isopropanol. Incubate on a roller at room temperature for 5 minutes.
Place the tube on a magnet and let the Dynabeads magnetic beads collect at the magnet for 3 minutes (or until the supernatant is clear). Remove the supernatant completely.
Remove the tube from the magnet and add 500 µl Washing Buffer 1. To wash, resuspend the Dynabeads magnetic beads thoroughly by pipetting.
Place the tube on a magnet and let the Dynabeads magnetic beads collect at the magnet for 2 minutes (or until the supernatant is clear). Remove the supernatant completely.
Remove the tube from the magnet and add 500 µl Washing Buffer 2. To wash, resuspend the Dynabeads magnetic beads thoroughly by pipetting.
Place the tube on a magnet and let the Dynabeads magnetic beads collect at the magnet for 2 minutes (or until the supernatant is clear). Remove the supernatant completely.
Let the pellet air-dry while still placed on the magnet (approx. 10 minutes).
Remove the tube from the magnet and add 100 µl Elution Buffer. Pipette until the pellet is completely resuspended. Incubate at room temperature for 2 minutes.
Place the tube on a magnet and let the Dynabeads magnetic beads collect at the magnet for 2 minutes (or until the supernatant is clear). Transfer the supernatant/eluate containing the purified total RNA to a fresh tube.
Add 10 µl 10x DNase Buffer to the eluates. Add 20 U DNase. Incubate at 37 degrees C for 10 minutes.

Answer Id: E6060

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Product FAQ

I am seeing high background following RNA hybridization. What could be causing this?

Answer

There are several types of background, and each can have a different cause:

1) Blotchy signal across the membrane:
This can be caused by a membrane of poor quality, one that has dried out, or one that has been mishandled (e.g., oil from human skin, powder from gloves). Use high quality nylon membrane that has not previously been handled and use forceps to handle the membrane from the edges. Blotchiness can also be caused by uneven distribution of the hybridization reagents. Do not pipette probe directly onto the membrane in hybridization solution; dilute it into the hybridization solution first.

2) A smear through the lane:
Hybridization conditions that are substantially below the optimum for a given probe can lead to high lane-specific background and/or substantial cross-hybridization. Start with a high hybridization temperature and slowly decrease the temperature until a specific signal is obtained. High probe concentrations, especially for nonisotopic probes, can also cause lane-specific background. Use 10 pM nonisotopically labeled DNA probes and 0.1 nM nonisotopically labeled RNA probes.

3) Speckling across the membrane:
Probe preparations with poor incorporation (or where unincorporated nucleotides have not been removed) can cause speckling on the membrane. Check probe quality and remove unincorporated nucleotides. Particulates in probe preparations or hybridization buffer (e.g., when not completely in solution) can also cause speckling on the membrane. Ensure that these reagents are in solution, and consider centrifuging in a microfuge or low-speed centrifuge, or filtering the solutions through a 0.22 µm filter to remove particulates.

If you see high background that is not associated with the lanes, this could be due to:

  • A bad membrane or incompatible membrane.
  • A membrane that dried out during procedure.
  • Reagents that were not evenly distributed.
  • Microbial contamination.
  • Particulate matter deposited on the membrane.
  • Precipitates present in nonisotopic detection reagents.
  • Agarose or transfer buffer that dried on the membrane.
  • Static charges developing during film development.
  • A blot that was too wet when exposed to film.

    Answer Id: E8036

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  • Product FAQ

    What are the elution conditions for immunoprecipitation using Dynabeads magnetic beads?

    Answer

    The elution method may either be mild or denaturing. Mild elution methods can typically be used and these include change of pH (low pH is most frequently used) and change of ionic strength (high salt concentration buffers (e.g. NaI, KI, MgCl, KCl)). Below you will find protocols for mild elution methods. Please note that the protocols may need to be optimized depending on the antibody's affinity to the target. Dynabeads magnetic beads coated with antibody may be reused several times by employing mild elution methods such as low pH or high salt. The success of elution depends on the strength of the interaction between the immobilized antibody and the captured target. In addition, the reusability of the immobilized antibody (when employing low pH treatments) also depends on sensitivity of the immobilized antibody toward this type of treatment. In general, using high salt concentration as the elution method assures several rounds of bead reuse; eluting with low pH typically results in yield decreases after a couple of rounds of reuse.

    Elution using low pH
    You can elute by lowering pH using 0.1 M citrate (pH 2-3) as the elution buffer. The degree of acidity needed depends on the species and IgG subclass, but at pH 3.1 most targets will be eluted.
    (1) Add 30 µL 0.1 M citrate to Dynabeads-target complex.
    (2) Mix well by tilting and rotation 2 min.
    (3) Place the test tube in a DynaMag magnet, and transfer the supernatant, containing purified target, to a clean tube.
    (4) Add additional 30 µL 0.1 M citrate to the Dynabeads magnetic beads to elute any remaining target.
    (5) Mix well by tilting and rotation 2 min.
    (6) Place the test tube in a DynaMag magnet, remove the eluate with a pipette and pool the supernatants containing pure target.

    Total collected volume = 60 µL

    Example of 0.1 M Citrate buffer, pH 3.1:
    (1) 10.5 g citric acid monohydrate
    (2) Distilled H2O to 450 mL
    (3) Adjust pH to 3.1 with 5 M NaOH
    (4) Distilled H2O to 500 mL

    Elution by change in ionic strength
    A buffer with an increasing salt concentration (e.g., 2 M NaI) is used to elute isolated protein.
    (1) Resuspend washed pellet of Dynabeads magnetic beads in 0.5 mL 2M NaI.
    (2) Mix vigorously on a whirlimixer/ vortexer for 1-2 min.
    (3) Place tube in DynaMag magnet for one minute then carefully remove supernatant to a clean tube.
    (4) Repeat point 1-3 two times and pool the supernatants.

    Direct elution of proteins into SDS-PAGE sample loading buffer (denaturing elution)
    (1) Resuspend washed pellet of antigen-bound Dynabeads magnetic beads in SDS-PAGE sample loading buffer.
    (2) Heat to 100 degrees C for 3 min.
    (3) Place tube in DynaMag magnet. Wait one minute then carefully remove supernatant for loading onto electrophoresis gel.

    Answer Id: E6017

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    Product FAQ

    How should I dilute my test compound for a drug discovery assay?

    Answer

    We recommend making a 100X serial dilution of the test compound in 100% DMSO solution. Here is the protocol:

    - Begin with a 1 mM stock solution of the test compound in 100% DMSO.
    - - This is the 100X in 100% DMSO starting point.
    - - This is appropriate for the highest 1X starting concentration of 10 µM.

    - In a DMSO-tolerant assay plate, place 20 µL of 100% DMSO in 9 wells, A2-A10, skipping well A1.

    - In well A1, place 30 µL of the 1 mM stock solution.

    - Transfer 10 µL of material from well A1 to A2, mix by pipette.

    - Repeat until there are 10 concentrations, discard the last 10 µL.

    Prepare an intermediate dilution of the test compound in assay buffer. This is typically done at 2X, 3X or 4X. Please refer to your specific assay protocol.

    Answer Id: E16540

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    Product FAQ

    What causes the appearance of diffuse bands and swirling patterns on membranes after Western transfer?

    Answer

    The swirling and diffuse banding patterns are typical of molecules moving laterally before binding to the membrane during transfer. Three possible explanations are:

    1) Poor contact between the gel and the membrane: the gel should contact the membrane across the entire surface for good capillary action. To ensure good contact and remove any bubbles, roll a glass pipette over the surface of each layer of the gel/membrane sandwich. Pipet some extra transfer buffer on the surface of each layer as the sandwich is being made. Also, the blotting pads need to be fully saturated with transfer buffer (push down with a gloved hand when they are submerged to ensure that there are no air bubbles).

    2) Undercompression of the gel: The gel/membrane assembly should be held securely between the two halves of the blot module. Try adding another pad or replace any pads that have lost their resiliency with new ones.

    3) Overcompression of the gel: A good indication of overcompression is if the gel has been excessively flattened. In the event that the sandwich is overcompressed, remove enough pads so that the blot module can be closed without exerting excess pressure on the gel and membrane. NOTE: The height of the uncompressed pads should be 0.5-1.0 cm above the level of the sealing gasket.

    Answer Id: E3284

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    Product FAQ

    How does one prepare serum and plasma samples?

    Answer

    Serum is the liquid fraction of whole blood that is collected after the blood is allowed to clot. The clot is removed by centrifugation and the resulting supernatant, designated serum, is carefully removed using a Pasteur pipette. Plasma is produced when whole blood is collected in tubes that are treated with an anti-coagulant. The blood does not clot in the plasma tube. The cells are removed by centrifugation. The supernatant, designated plasma, is carefully removed from the cell pellet using a Pasteur pipette.

    Serum preparation: Collect whole blood in a covered test tube. If commercially available tubes are to be used, the researcher should use the red topped tubes. These are Becton Dickinson Vacutainer tubes. After collection of the whole blood, allow the blood to clot by leaving it undisturbed at room temperature. This usually takes 15-30 minutes. Remove the clot by centrifuging at 1,000-2,000 x g for 10 minutes in a refrigerated centrifuge. The resulting supernatant is designated serum. Following centrifugation, it is important to immediately transfer the liquid component (=serum) into a clean polypropylene tube using a Pasteur pipette. The samples should be maintained at 2-8°C while handling. If the serum is not analyzed immediately, the serum should be apportioned into 0.5 mL aliquots and stored and transported at -20°C or lower. It is important to avoid freeze/thaw cycles because this is detrimental to many serum components. Samples which are hemolyzed, icteric, or lipemic can invalidate certain tests.

    Plasma preparation: Collect whole blood into commercially available anti-coagulant treated tube, such as EDTA treated (lavender tops) or citrate treated (light blue tops). Heparinized tubes (green tops) are indicted for some applications; however, heparin can often be contaminated with endotoxin and endotoxin can stimulate white blood cells to release cytokines. Cells are removed from plasma by centrifugation for 10 minutes at 1,000-2,000 x g using a refrigerated centrifuge. Centrifugation for 15 minutes at 2,000 x g depletes platelets in the plasma sample. The resulting supernatant is designated plasma. Following centrifugation, it is important to immediately transfer the liquid component (=plasma) into a clean polypropylene tube using a Pasteur pipette. The samples should be maintained at 2-8°C while handling. If the plasma is not analyzed immediately, the plasma should be apportioned into 0.5 mL aliquots and stored and transported at -20°C, or lower. It is important to avoid freeze/thaw cycles. Samples which are hemolyzed, icteric, or lipemic can invalidate certain tests.

    There are other commercially available tubes for blood sample collection. Thermo Fisher Scientific has not evaluated some of these tubes for compatibility with our ELISA kits. The commercially available serum tubes are as follows:Red: No anticoagulant. Red with black: treated with gel to help to separate the clot (not evaluated). The commercially available plasma tubes are as follows: Lavender: Treated with EDTA. Blue: Treated with citrate. Green: Treated with heparin. Grey: Treated with potassium oxalate/sodium fluoride (not evaluated). Yellow: Treated with ACD (not evaluated).

    References: 1. Henry, J.B. (1979) Clinical Diagnosis and Management by Laboratory Methods, Volume 1, W.B Saunders Company, Philadelphia, PA, p. 60. 2. Thavasu, P.W., S. Longhurst, S.P. Joel, M.L. Slevin, and F.R. Balkwill (1992) Measuring cytokine levels in blood. Importance of anticoagulants, processing, and storage conditions. J. Immunol. Methods 153:115-124.

    Answer Id: E5129

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