Posts

PCR [Polymerase Chain Reaction]

Image
Polymerase Chain Reaction (PCR) Source PCR was developed by Kary Mullis in 1983. It utilizes Taq DNA polymerase , an enzyme derived from the thermophilic bacterium Thermus aquaticus , which remains active at high temperatures required for DNA denaturation. Principle PCR is a technique used to amplify specific DNA sequences. It involves repeated cycles of denaturation, annealing, and extension. Denaturation : Heat (94–98°C) separates the double-stranded DNA into single strands. Annealing : Cooling (50–65°C) allows primers to bind to complementary sequences on the target DNA. Extension : DNA polymerase synthesizes a new DNA strand by adding nucleotides to the primers at ~72°C. Instrumentation Thermal Cycler : Programmable machine to perform temperature cycling for denaturation, annealing, and extension. Reagents : Template DNA : The target DNA to be amplified. Primers : Short oligonucleotides complementary to the target sequence. dNTPs : Building blocks for DNA synthesis. Taq Polymerase ...
Post a Comment
Read more

Plaque Blotting

Image
Plaque Blotting Principle Plaque blotting is a technique used to detect specific DNA or RNA sequences in bacteriophage plaques. Phage DNA or RNA is transferred from agar plates to a membrane, lysed, denatured, and hybridized with a labeled probe to identify target sequences. Instrumentation Agar Plates with Phage Plaques : Plaques formed by bacteriophage-infected bacterial lawns. Membrane : Nylon or nitrocellulose to capture phage nucleic acids. Vacuum or Capillary Blotting Apparatus : For transferring nucleic acids to the membrane. Hybridization Oven : Ensures uniform temperature for probe hybridization. Detection System : Autoradiography for radioactive probes. Chemiluminescence or colorimetric systems for non-radioactive probes. Types of Plaque Blotting DNA Plaque Blotting : Detects specific DNA sequences in phage plaques. RNA Plaque Blotting : Identifies RNA transcripts associated with phage DNA. Applications of Plaque Blotting Library Screening : Identifying clones in phage displa...
Post a Comment
Read more

Colony Blotting

Image
Colony Blotting Principle Colony blotting is a technique used to detect specific DNA or RNA sequences in bacterial or yeast colonies. Colonies grown on agar plates are transferred onto a membrane, lysed to release nucleic acids, and hybridized with a labeled probe to identify the target sequence. Instrumentation Agar Plates : Contain bacterial or yeast colonies. Membrane : Nylon or nitrocellulose to capture DNA/RNA from colonies. Hybridization Oven : Maintains optimal conditions for probe hybridization. Detection System : Autoradiography for radioactive probes. Chemiluminescence or colorimetric detection for non-radioactive probes. Types of Colony Blotting DNA Colony Blotting : Detects specific DNA sequences in colonies. RNA Colony Blotting : Identifies RNA transcripts within colonies. Protein Colony Blotting : Uses antibodies to detect proteins directly from colonies. Applications of Colony Blotting Gene Identification : Screening bacterial or yeast colonies for specific genes. Recomb...
Post a Comment
Read more

Western Blotting

Image
Western Blotting Principle Western blotting detects specific proteins in a sample using a combination of gel electrophoresis, transfer to a membrane, and antibody-based detection. It relies on the specificity of antibodies to target proteins, enabling quantitative or qualitative analysis. Instrumentation SDS-PAGE Apparatus : For separating proteins based on molecular weight. Blotting Apparatus : Transfers proteins from gel to membrane (electroblotting or semi-dry blotting systems). Membrane : Nitrocellulose or polyvinylidene difluoride (PVDF) for protein immobilization. Blocking and Hybridization Setup : Trays or incubators for applying blocking agents, primary antibodies, and secondary antibodies. Detection Systems : Chemiluminescent imaging systems. Colorimetric detection. Fluorescence-based detection. Types of Western Blotting Conventional Western Blotting : Uses chemiluminescent or colorimetric detection systems with enzyme-linked antibodies. Fluorescent Western Blotting : Utilizes...
Post a Comment
Read more

Principle, instrumentation, types of Dot blotting

Image
Dot Blotting Principle Dot blotting is a rapid technique for detecting specific DNA, RNA, or proteins. The sample is directly applied as a dot onto a membrane without electrophoresis. Hybridization or antibody binding with a labeled probe enables specific detection. Instrumentation Membrane : Nylon or nitrocellulose to immobilize the sample. Dot Blot Apparatus or Template : Ensures uniform application of the sample. Hybridization Oven : Maintains controlled conditions for probe binding. Detection System : Autoradiography for radioactive probes. Chemiluminescence or colorimetric systems for non-radioactive probes. Types of Dot Blotting DNA Dot Blotting : Detects specific DNA sequences using labeled probes. RNA Dot Blotting : Identifies RNA transcripts through hybridization with complementary probes. Protein Dot Blotting : Detects specific proteins using antibodies. Applications of Dot Blotting Gene Expression Analysis : Screening for the presence of specific DNA or RNA in a sample. Prot...
Post a Comment
Read more

Northern Blotting

Image
Northern Blotting Principle Northern blotting is used to detect specific RNA sequences by hybridizing them with a complementary labeled probe after separating RNA by size using denaturing gel electrophoresis. It allows for the study of gene expression and RNA processing. Instrumentation Gel Electrophoresis Unit : Separates RNA based on size. Denaturing Agarose Gel : Prevents RNA secondary structures during separation. Blotting Apparatus : Transfers RNA from gel to membrane (capillary or vacuum transfer). Membrane : Nylon or nitrocellulose membrane for RNA immobilization. Hybridization Oven : Maintains optimal temperature for probe binding. Detection System : Autoradiography for radioactive probes. Chemiluminescent or fluorescent systems for non-radioactive probes. Types of Northern Blotting Traditional Northern Blotting : Uses radioactive probes for sensitive RNA detection. Non-Radioactive Northern Blotting : Safer probes such as digoxigenin or biotin-labeled probes. Quantitative North...
Post a Comment
Read more