Source: Molecular Cytogenetic Technology and Applications
Operation method
basic program
Principle
The Universal Linkage System (ULS®; KREATECH Biotechnology BV) is a method of labeling with a platinum-stained complex that reacts with the N7 residue of a guanine nucleoside. The reaction results in the formation of a stable chemical bond between the nucleic acid and the platinum fluorescent group. Depending on the reaction conditions, ULS compounds also form complexes with adenine to a lesser extent. The method has been used for DNA (including plasmids, mucilages, molecular mass references, DNA in low melting point agarose, BAC, PAC, YAC, whole-chromosome libraries, DOP-PCR products such as highly repetitive sequences of satellites, filaments, and telomeric DNAs), RNA, PNA, oligonucleotides, and amplified nucleic acid products. ULS reagents and kits are likewise available at Molecular Probes (Eugene, OR). The size of the template for labeling with any ULS compound must be less than 1000 bp. templates larger than 1000 bp usually produce a large amount of dotted background. pcR products are generally less than 1000 bp and can therefore be labeled directly. The two methods of DNA fragmentation described below, sonication and digestion, can be used in both the Kreatech and Molecular Probes procedures.Another DNAzyme I procedure is recommended for Molecular Probes when swabbing with Alexa-ULS. The procedure and corresponding reagents are part of the ULS labeling kit provided by Molecular Probes.
Materials and Instruments
DNA samples Move 1 DNA fragmentation 1.1 Ultrasonic fragmentation 1. Prepare a DNA solution (20ng/μL) with TE, preferably with at least 100μL of DNA solution for sonication. 2. Place the sample on ice and use a small conical-bottomed plastic tube to ultrasonically crush the DNA in 3 cycles of 1 min each. select the power level and number of cycles of the ultrasonic crusher, trying to use the highest possible power with as little vacuole formation as possible. The DNA solution was pre-cooled on ice for 1 min before fragmentation and after each cycle. Centrifuge the DNA solution at the highest speed of the microcentrifuge for 5s before the second and third cycles so that all the solution falls to the bottom of the tube. 3. A portion of the DNA solution was taken and electrophoresed on a 1% agarose gel to determine if the DNA was the right size. 4. Start the labeling procedure. 1.2 DNAzyme processing 1. Prepare a storage solution for DNAzyme I: Dissolve 1 mg of DNAzyme I (approximately 2000 U/mg, Roche) with 1 mL of 5 mmol/L sodium acetate, 1 mmol/LCaCl2, 50% glycerol pH 5.2. The buffer was placed on ice before and during the addition of lyophilized DNAzyme. The solution was turned up and down until completely mixed without shaking, and the storage solution was kept at -20°C and avoided repeated freezing and thawing as much as possible. 2. Dilute the DNAzyme stock solution with 1X nick-panning buffer at a ratio of 1:500. 3. Add the following components to a small centrifuge tube on ice: 1 μL of template DNA, 2.5 μL of 10X Notch Pan Buffer, 3~5 μL of diluted DNAzyme I, and add water to 25 μL. 4. Place the small centrifuge tube at 37℃ for 10min. 5. Place the small centrifuge tube on ice to terminate the reaction. 6. Add 1/4 volume of 10 mmol/L ammonium acetate and 2.5 times volume of anhydrous ethanol to the above reaction for precipitation. 7. Resuspend the precipitate with an appropriate volume of labeling solution. 8. take a portion of the DNA solution and electrophoresis it on a 1% agarose gel to determine if the DNA is the right size 2 Labeling 2.1 KREATECH recommended procedure Suitable labeling efficiency is obtained when ULS reagent is mixed with nucleic acid in a 1:1 ratio (e.g., IUULS reagent: 1 μg of DNA). When the amount of labeled nucleic acid is not the standard 1 μg, the ratio of nucleic acid to ULS reagent must be guaranteed to be 1:1. when labeling a small amount of nucleic acid, it should be at least 100 ng, 20 μL volume. Also, labeling larger amounts of nucleic acid should not exceed 10 μg of nucleic acid in 20 μL. Only template concentrations not less than 5ng/μL can be labeled in larger volumes, and the amount of ULS reagent is adjusted with the amount of template added. If the template dilution is too large, the labeling solution can be left out. 1. Add 1U (2μL) of ULS reagent to 1μg of template. 2. Adjust the volume to 20μL with labeling solution and mix well. 3. incubate at 65℃ for 15min. 4. Centrifuge slightly. 5. Purify the probe by centrifugation column. 2.2 Recommended Procedures by Molecular Probes, Inc. Molecular Probes offers a kit for the application of the ULS labeling system in combination with various dyes. The kit includes a variety of reagents for fragmenting DNA and labeling probes. The labeling procedures and precautions are basically the same except for the following. 1. Prior to labeling, Molecular Probes' ULS dye reagents are dissolved in different buffers depending on the dye selected. A chart is available that provides all the specific information associated with each dye. 2. Prior to labeling, the DNA is denatured at 95°C for 5 min and then rapidly cooled on ice. Please note: denaturation is not a required step, but can improve labeling efficiency by 20% to 40%. 3. The volume of labeling reagent is 25μL. 4. Incubate at 80℃ for 15min. 5. MolecularProbes states that the procedure previously provided by Kreatech has been modified due to the specificity of the company's proprietary dyes bound to the ULS reagent. Caveat 1. When ultrasonic crushing, the sample volume, the type of container containing the sample, the ultrasonic crushing time and number of cycles, the power level of the ultrasonic crusher and the working cycles all vary according to the ultrasonic crusher manufacturer, model and probe. Pre-experimentation is sometimes necessary to obtain nucleic acid fragments of the right size. 2. All reagents should be placed on ice when performing DNAzyme I digestion. Prepare the solution temporarily before use. 3. DNA should be purified to remove proteins, RNA and free nucleotides before labeling. 4. High concentrations of Tris (>40 mmol/L) or EDTA (﹥5 mmol/L), magnesium acetate (>100 mmol/L), NaCl (>100 mmol/L) and restriction endonuclease digestion buffers should be avoided as much as possible because of the rate-limiting effect of these on the labeling reaction. For more product details, please visit Aladdin Scientific website.