Labeling experiments with DNA probes
Labeling experiments with DNA probes
Nucleic acid probe molecular hybridization refers to the two nucleic acid single strands with certain homology can be formed under certain conditions according to the principle of base complementarity double strand, this hybridization process is highly specific. Nucleic acid probes can be divided into DNA and RNA probes according to the nature of nucleic acids; according to the different markers, they can be divided into two categories: radiolabeled probes and non-radiolabeled probes; according to the existence of complementary chains, they can be divided into single-stranded and double-stranded probes; according to the radiolabel incorporation, they can be divided into uniformly labeled and end-labeled probes.
Operation method
probe labeling method
Principle
Molecular hybridization, a way of binding nucleic acid strands with base-pairing rules, is an important physicochemical property of nucleic acids. To use this property of molecular hybridization to detect specific nucleic acid sequences, one of the hybridized chains must be labeled with some detectable, which is called a nucleic acid probe. Therefore, the preparation of nucleic acid probes is the key to molecular hybridization technology. Radioisotope labeling is the earliest and currently the most commonly used method of labeling nucleic acid probes.
Materials and Instruments
EGFR-PCR product washing buffer Detection buffer TE buffe Move 1. Dilute 1 ug DNA in sterilized deionized water to a total volume of 16 ul. Caveat 1. DNA template for labeling is purified as much as possible to improve labeling efficiency2. Template DNA >100 bp.3. DIG labeled probes cannot be purified by phenol/chloroform extraction.4. Determine labeling efficiency according to the instructions (labeled probe and kit positive control DIG-labeled DNA gradient dilution, nylon membrane spotting, sample denaturation, blocking, hybridization, colorimetric quantification).5. This method can also be used for single-stranded DNA or RNA probe labeling, when the labeling of RNA probe, the need to use reverse transcriptase, the harvested product is labeled single-stranded cDNA.6. The product can be labeled according to the following table. For more product details, please visit Aladdin Scientific website.
EGFR-PCR product washing buffer Detection buffer TE buffe
eppendorf tubes Tip head PCR instrument Tweezers Powder-free gloves Volume sampler
2. Thermal denaturation of DNA: DNA samples were denatured at 100°C for 10 min in a PCR instrument and then quickly placed in crushed ice for more than 3 min.
3. Add 4 ul DIG-Random Labeling Mix, mix well and centrifuge at 2000 rpm × 5 min (4°C).
4. The PCR instrument was reacted at 37°C overnight.
5. Add 2 ul EDTA to terminate the reaction and the labeling is finished. Store the marker at -20°C for >1 year
