Label it® nucleic acid labeling kit, cx-rhodamine protocol

Protocol
Page 1 of 6 Lit.# ML002 Rev. 10/04/12
Label IT® Nucleic Acid Labeling Kits Product Name
Quantity*
Product No.
Label IT® CX-Rhodamine Labeling Kit Label IT® Fluorescein Labeling Kit Label IT® TM-Rhodamine Labeling Kit *Each Full Size Kit contains sufficient reagents to Label 100 µg of nucleic acid. Each Trial Size Kit contains sufficient reagents to Label 25 µg of nucleic acid. 1.0 DESCRIPTION
1.1 General Information
Mirus' Label IT Nucleic Acid Labeling Technology represents a new class of labeling reagents designed to efficiently and reproducibly attach marker molecules to nucleic acids (DNA or RNA) in a simple one-step reaction within minutes. The Label IT Nucleic Acid Labeling Reagents covalently attach CX-rhodamine, fluorescein, digoxin, biotin, Cy™3, Cy™5, dinitrophenol (DNP), and TM-rhodamine labels to nucleic acid bases within DNA or RNA without impacting hybridization performance, and therefore can be used in almost any molecular biology application (excitation and emission wavelengths for each fluorescent Label IT Reagent are listed in Table 1). The simplicity of the Labeling reaction, and the fact that only a single reagent is required, suggests that this technology can be widely used in a variety of nucleic acid Labeling applications. Traditional nonradioactive Labeling methods (random priming, nick translation) are enzyme mediated and thus inherently difficult to control. In addition, these types of reactions generate Labeled products that are not representative of the starting nucleic acid but rather consist of a series of Labeled samples over a variable size range. The Labeling efficiency of these reactions is dictated by the enzyme's ability to incorporate a "Labeled-nucleotide" precursor into a growing nucleic acid chain. This Labeled-nucleotide is not the preferred substrate for the enzyme and may compromise the efficiency of the reaction and introduce a Labeling bias. In contrast, the Label IT Labeling reactions are nondestructive, easy to control, and can be scaled up or down by either the size of the reaction or the desired Labeling density. The supplied standard Labeling protocol will yield Labeling efficiencies of approximately one Label every 20-60 base pairs of double-stranded DNA. Mirus has found that this Labeling density is sufficient to allow sensitive detection for the majority of applications. If there is a need to increase or decrease the density of Labels in the final product, simply modify the ratio of Labeling reagent to nucleic acid during the Labeling reaction or adjust the incubation time of the labeling reaction. 545 Science Dr. • Madison, WI 53711 USA • Ph. 888.530.0801, or 608.441.2852 • Fax. 608.441.2849 • www. mirusbio.com Protocol
Page 2 of 6 Lit.# ML002 Rev. 10/04/12


Table 1. Excitation and emission wavelengths For Fluorescent Label
IT Reagents
Fluorophore
Excitation Wavelength (nm)
Emission Wavelength (nm)
1.2 Materials Supplied

Reagent Cap
Kit Component*
Full Size
Trial Size
*Extra volume of each kit component is supplied to allow for differences in pipetting devices. 1.3 Materials Required but Not Supplied
If required, detection reagents, including conjugated antibodies, conjugated streptavidin, and chemiluminescent substrates, can be obtained from a variety of commercial sources. 1.4 Storage and Stability
Store the Label IT Reagent at –20°C in both its dried pellet and reconstituted form. Cap the Label IT Reagent tightly and
avoid exposure to moisture and light. Store all other supplied reagents, including the G50 microspin purification
columns, at 4°C. Do not use the G50 microspin columns if they have been stored at –20°C or colder. The Label IT
Reagent is stable for 6 months after reconstitution. Unreconstituted Label IT Reagent and all other reagents are stable for
up to 1 year from the date of purchase.
2.0 PROCEDURE
2.1 Labeling Reaction
1. Prior to each use, warm the vial containing the Label IT Reagent to room temperature and quick spin to collect the
pellet. If using a full size kit, add 100 µl of Reconstitution Solution to the pellet in the vial. If using a trial size kit, add 25 µl of the Reconstitution Solution to the pellet in the vial. To ensure complete reconstitution of the pellet, mix well by gentle pipetting, then perform a quick spin. 2. Prepare the Labeling reaction according to the example shown below. Use water that is both RNase- and DNase- free (molecular biology-grade quality). Add the Label IT Reagent last. Use only purified nucleic acid (A260/A280 between 1.8 and 2.2) in the Labeling reactions. Labeling Reaction Example: Molecular biology-grade H2O 35 µl 10X Labeling Buffer A 1 mg/ml nucleic acid sample 5 µl Label IT Reagent Total volume: 50 µl
545 Science Dr. • Madison, WI 53711 USA • Ph. 888.530.0801, or 608.441.2852 • Fax. 608.441.2849 • www. mirusbio.com Protocol
Page 3 of 6 Lit.# ML002 Rev. 10/04/12
NOTE: This example Labels 5 µg of nucleic acid at a 1:1 (v:w) ratio of Label IT Reagent to nucleic acid. This ratio
will result in Labeling efficiencies that are appropriate for most applications. If there is a need to increase or
decrease the density of Labels in the final product, simply modify the ratio of Labeling reagent to nucleic acid
during the Labeling reaction or adjust the incubation time of the Labeling reaction. In addition, the Labeling
reaction may be scaled up or down, depending on the amount/volume of nucleic acid to be Labeled. When scaling
the Labeling reaction, the amount of Label IT Reagent should never constitute more than 20% of the total reaction
volume. Ensure that the final concentration of Labeling Buffer A is 1X.
3. Incubate the reaction at 37°C for 1 hour. A quick spin should be performed after 30 minutes of incubation. This will minimize the effect of evaporation and maintain the appropriate concentration of the reaction components.
NOTE: To denature Labeled DNA for a hybridization application, please see Application Notes (Section 3.0, Part
B).
4. Purify the Labeled sample by either ethanol precipitation (see Section 2.2) or using the provided G50 Microspin Purification Columns (see Sections 2.3). Microspin column purification may be particularly beneficial for purification of small amounts (under 1 µg) of nucleic acid. These columns, however, are not recommended if the Labeled sample needs to be quantified by spectrophotometry as gel filtration columns can lead to erroneously high ultraviolet absorbance readings. 2.2 Purification using Microspin Columns
NOTE: The microspin columns are not recommended for nucleic acids under 20 bases in length. Do not use the
microspin columns if it is necessary to quantify the Labeled sample after purification. It is generally acceptable to
assume 100% recovery of the Labeled nucleic acid following microspin column purification. If the exact quantity
of the purified Labeled sample is imperative, use an alternate purification procedure (see Section 2.3, Purification
using Ethanol Precipitation).
NOTE: The reaction volume applied to the G50 Microspin Purification Column must be 50 µl. If the reaction
volume is less than 50 µl, add 1X Labeling Buffer A to bring the volume to 50 µl. If the reaction volume exceeds
50 µl, split the reaction volume and use 50 µl per column.
A. Centrifuge
Conditions
Before using a microspin column, it is important to calculate the speed at which the column should be centrifuged. For a force of 735 x g, the appropriate speed can be calculated from the following formula: where r = radius (in mm) measured from the center of spindle to bottom of the rotor bucket and rpm = revolutions per minute. For example, with a rotor having a radius of 73 mm, the appropriate speed would be 3,000 rpm. B. Column Preparation
1. Vortex to resuspend the resin in the column.
2. Loosen the cap one-fourth turn then pull out the bottom closure.
3. Place the column in a 1.5 ml screw-cap microcentrifuge tube for support. Alternatively, remove the cap from a flip-
top microcentrifuge tube and use this tube as a support. 4. Spin the column for 1 minute at 735 x g (e.g., 3000 rpm in an Eppendorf 5415C variable-speed centrifuge with an 18-position fixed-angle rotor; see Section A above). Start the timer and microcentrifuge simultaneously.
NOTE: Do not pulse-spin, as this will override the variable speed setting. Use columns immediately after
preparation to prevent dehydration of the resin.
5. Discard buffer collected during spin. C. Sample Application
1. Place the column in a new 1.5 ml microcentrifuge tube.
2. Slowly apply the sample (50 µl) to the top center of the resin. Do not disturb the resin bed.
3. Spin the column at 735 x g for 2 minutes. The purified sample will collect in the bottom of the support tube.
4. Cap the support tube. The Labeled sample is now ready for use. See Section 3.0 for application suggestions.
545 Science Dr. • Madison, WI 53711 USA • Ph. 888.530.0801, or 608.441.2852 • Fax. 608.441.2849 • www. mirusbio.com Protocol
Page 4 of 6 Lit.# ML002 Rev. 10/04/12
2.3 Purification using Ethanol Precipitation
NOTE: This purification method is optimal if quantification of the Labeled nucleic acid is necessary. For best
recoveries, increase the total volume to 200 µl with 1X Mirus Labeling Buffer A or molecular biology-grade H2O
before adding the sodium chloride and ethanol.
1. If purifying Labeled DNA, add 0.1 volume of 5 M sodium chloride then 2 volumes of ice cold 100% ethanol to the Labeling reaction. Mix and place at –20°C (or colder) for at least 30 minutes. If purifying Labeled RNA, add 0.1 volume of 5 M sodium chloride then 2.5-3 volumes of ice cold 100% ethanol to the Labeling reaction. Mix and place at –20°C (or colder) for at least 30 minutes. 2. Centrifuge at full speed in a microcentrifuge for at least 10 minutes to pellet the Labeled nucleic acid. Aspirate the ethanol, being careful not to disturb the pellet.
NOTE: Orient the tubes in the microcentrifuge in such a way that it is known where the pellet forms. Small nucleic
acid quantities can be invisible to the naked eye.
3. Gently wash the pellet once with 400-500 µl RNase- and DNase-free 70% ethanol (room temperature). 4. After an additional centrifugation (4°C) at full speed for 10 minutes, remove all traces of ethanol with a micropipetter. Do not allow the sample to air dry extensively, as the pellet may become difficult to resuspend. 5. Resuspend the Labeled nucleic acid in 1X Labeling Buffer A or the buffer required for the particular application. 3.0 APPLICATION NOTES

A. Non-Radioactive Hybridization Applications
Conjugated antibodies, conjugated streptavidin, chemiluminescent substrates, and other reagents for biotin, digoxin, DNP and fluorescein detection can be obtained from a variety of sources. For non-radioactive DNA-based membrane hybridization applications, the HybQUEST® Hybridization and Detection Kit (see Related Products, Section 5.0) contains the reagents needed for the preparation, hybridization, and detection of DNP-Labeled samples. B. Hybridization Reactions using Labeled DNA Samples
For optimal sensitivity and stability of the Labeled DNA sample in hybridization reactions, use the supplied Denaturation Reagent D1 and Neutralization Buffer N1 to denature the Labeled sample prior to any hybridization applications. Do not heat-denature the Labeled DNA probe before this step. Once treated with Denaturation Reagent D1 and Neutralization Buffer N1, Labeled DNA samples can be heat denatured as required. 1.Just prior to the hybridization, add 0.1 volume of Denaturation Reagent D1 to the Labeled sample. Mix well and incubate for 5 minutes at room temperature. 2. Add 0.1 volume of Neutralization Buffer N1. Mix well and incubate on ice for at least 5 minutes. The Labeled sample is now ready to be used in any hybridization protocol. If the denatured sample is to be used at a later time, store at –20°C and avoid multiple freeze/thaws to maintain the denatured state. C. Hybridization Reactions using Labeled RNA Samples
For optimal sensitivity and stability of the Labeled RNA probe, denature the RNA by heating at 55-65°C for 10 minutes prior to any hybridization applications. Do not denature the Labeled RNA probe with Denaturation Reagent D1 and Neutralization Buffer N1, as alkaline conditions can hydrolize RNA. D. Fluorescent In Situ Hybridizations
The Label IT technology is ideal for use in ISH/FISH applications. Mirus has designed the Label IT FISH Kits (see
Related Products, Section 5.0) to vividly paint chromosomes or centromeres and reveal the location of specific
DNA sequences. The optimized
Label IT FISH Kits utilize Mirus’ Label IT technology to prepare superior Labeled probes for hybridization to
interphase/metaphase chromosome spreads.
E. Expression Profile Analysis on Microarrays
The Label IT µArrayBiotin Labeling Kits (see Related Products, Section 5.0) are designed to directly Label mRNA, cDNA, or cRNA for expression profile analysis in microarray applications. These kits provide robust performance demonstrated by high signal to noise ratios, consistent replicates and sensitive detection. 545 Science Dr. • Madison, WI 53711 USA • Ph. 888.530.0801, or 608.441.2852 • Fax. 608.441.2849 • www. mirusbio.com Protocol
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F. In Vitro Tracking Experiments
Mirus has developed Label IT Tracker™ (DNA tracking) and Label IT siRNA Tracker (siRNA tracking) Intracellular Localization Kits (see Related Products, Section 5.0) that provide the necessary reagents to directly Label and transfect either plasmid DNA or siRNA,in an efficient and nondestructive manner. Both subcellular localization and functionality can be monitored in the same experiment following the delivery of the Labeled sample into mammalian cells in culture. G. In Vivo Tracking Experiments
Both subcellular localization and reporter transgene expression can be monitored following the introduction of Labeled plasmid DNA into mammalian cells in vivo. Efficient in vivo delivery of Labeled DNA can be achieved using Mirus' TransIT® In Vivo Gene Delivery System (see Related Products, Section 5.0). This kit is designed for the non-viral delivery of transgenes into laboratory animals via tail vein injection. This kit primarily targets the liver, with lower levels of expression detected in the spleen, lung, heart, and kidneys. 4.0 TROUBLESHOOTING
Suboptimal Nucleic Acid Labeling
• Poor quality of Nucleic Acid
Use purified nucleic acid (A260/A280 between 1.8 and 2.2) that is free from proteins, carbohydrates, etc. Avoid nucleic acid degradation by using DNase- and RNase-free handling procedures and plasticware. Incubate the reaction at 37°C for 1 hour. The reaction may be extended to 2 hours to increase the Labeling density. A quick spin after 30 minutes will minimize the effect of evaporation. • Insufficient volume of Label IT Reagent was added to the reaction Use 1 µl of Label IT Reagent per 1 µg of nucleic acid. See example in Section 2.1 for proper Labeling reaction setup. • Labeling reaction was not scaled properly Keep the volume of Label IT Reagent less than 20% of the total reaction volume. Avoid using nucleic acid samples in high salt; NaCl concentrations greater than 50 mM can inhibit the Labeling reaction. Ensure that the final concentration of Buffer A is 1X. Store both reconstituted and unreconstituted Label IT Reagent tightly capped at -20°C. Protect from exposure to light and moisture. Warm vial to room temperature before opening. • Microspin columns were not stored properly Store columns at 4°C. Do not freeze. Do not use if they have been frozen. • Nucleic acid pellets were allowed to overdry Do not allow the Labeled nucleic acid pellet to dry extensively after ethanol precipitation. Remove traces of the ethanol wash and resuspend immediately in 1X Buffer A or low salt buffer of choice. • Use of the Denaturation Reagent D1 and Neutralization Buffer N1 Labeled DNA samples intended for hybridization applications must be treated with Denaturation Reagent D1 and Neutralization Buffer N1 as described in the Application Notes (see Section 3.0). This procedure denatures the DNA and stabilizes the Label IT Labels. Labeled DNA samples treated with Denaturation Reagent D1 and Neutralization Buffer N1 can be heat denatured if required. Do not heat denature Labeled DNA before treating with Denaturation Reagent D1 and Neutralization Buffer N1. Do not denature Labeled RNA with Denaturation Reagent D1 and Neutralization Buffer N1, as alkaline conditions can destroy RNA. Determining the Density of Labels on the Nucleic Acid Sample
NOTE: The relative density of Labels on purified, Labeled nucleic acid can be estimated by one of the following
methods:
For fluorescent dyes:
1. Spectrophotometric absorbance at λmax of the dye. Several micrograms of purified Labeled sample may be
required to generate significant λmax absorbance readings (for further details, see Label IT Reagent Frequently Asked Questions at www.mirusbio.com). 545 Science Dr. • Madison, WI 53711 USA • Ph. 888.530.0801, or 608.441.2852 • Fax. 608.441.2849 • www. mirusbio.com Protocol
Page 6 of 6 Lit.# ML002 Rev. 10/04/12
2. Fluorescent microscopy. Spot serial dilutions of purified Labeled sample onto a glass slide and view with a
fluorescent microscope.
For non-fluorescent dyes:
1. Dot blot analysis. Fix dilutions of the Labeled sample to a membrane, then detect with appropriate reagents.
2. Gel shift analysis. A Labeled sample demonstrates a distinct reduction in electrophoretic mobility compared to
For specific questions or concerns, please contact Mirus’ Technical Support at 888.530.0801 or [email protected] For a list of citations using Mirus’ products, please visit the Technical Resources section of our website (www.mirusbio.com). 5.0 RELATED PRODUCTS
Label IT® Tracker™ Intracellular Nucleic Acid Localization Kit (Product # MIR 7010, 7011, 7012, 7013, 7014, 7015) Label IT® siRNA Tracker Intracellular Localization Kit (Product # MIR 7202, 7203, 7204, 7205) Label IT® µArrayBiotin Labeling Kits (Product # MIR 8010 and MIR 8050) HybQUEST® Complete DNP System (Product # MIR 6000) HybQUEST® Label IT® Kits (Product # MIR 6200, 6300, 6400, 6800) HybQUEST® Hybridization and Detection Kit (Product # MIR 6010) TransIT®-In Vivo Gene Delivery System (Product # MIR 5100) TransIT®-293 Transfection Reagent (Product # MIR 2700)
TransIT®-3T3 Transfection Kit (Product # MIR 2180)
TransIT®-CHO Transfection Kit (Product # MIR 2170)
TransIT®-COS Transfection Kit (Product # MIR 2190)
TransIT®-Express Transfection Reagent (Product # MIR 2000)
TransIT-HeLaMONSTER® Transfection Kit (Product # MIR 2900)
TransIT®-Insecta Transfection Reagent (Product # MIR 2200)
TransIT®-Jurkat Transfection Reagent (Product # MIR 2120)
TransIT®-Keratinocyte Transfection Reagent (Product # MIR 2800)
TransIT®-LT1 Transfection Reagent (Product # MIR 2300)
TransIT®-LT2 Transfection Reagent (Product # MIR 2400)
TransIT-Neural® Transfection Reagent (Product # MIR 2140)
TransIT®-Oligo Transfection Reagent (Product # MIR 2160)
TransIT®-Prostate Transfection Kit (Product # MIR 2130)
TransIT-TKO® siRNA Transfection Reagent (Product # MIR 2150)
For determination of gene expression efficiency: Beta-Gal Staining Kit (Product # MIR 2600) MiraCLEAN® Endotoxin Removal Kit (Product # 5900) *These products are available in additional sizes.
TransIT, TransIT-TKO, MiraCLEAN, HeLaMONSTER, TransIT-Neural, HybQUEST and Label IT are registered trademarks of Mirus Corporation. Label IT Tracker and Label IT µArray are trademarks of Mirus Corporation. Mirus Transfection Reagents are covered by United States Patent No. 5,744,335; 5,965,434; 6,180,784; 6,383,811 and patents pending. Label IT Reagents are covered by U.S. Patent No. 6,262,252 and 6,593,465. Cy3 and Cy5 are trademarks of GE Corp. Cy3 and Cy5 products or portions thereof are manufactured under license from Carnegie Mellon University and are covered by U.S. Patent No. 5,268,486. 2006, Mirus Bio Corporation. All rights reserved. 545 Science Dr. • Madison, WI 53711 USA • Ph. 888.530.0801, or 608.441.2852 • Fax. 608.441.2849 • www. mirusbio.com

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