Public Services in the Study and Analysis of Insect and Arthropod Venom
- Design and review of primer specificity and specificity
- Extraction of DNA, RNA and Protein
- Determination of concentration, purity and integrity of extracted nucleic acid
- Performing PCR Techniques:
- Real Time -PCR qualitative and quantitative
- RT – PCR
- Nasted – PCR
- PCR – RFLP
- Electrophoresis based on agarose gel and polyamide
- Gel Document by Gel Doc
- Perform SDS-PAGE and Western Blot
- Check the purity
- Check the amount of protein available
۱- Extraction of DNA from Microorganisms, Blood, Cell Lines, Fresh Tissue, Paraffin Tissue:
DNA extraction is performed by either phenol chloroform, salting out, or using commercial kits in the laboratory. In phenol chloroform method, first, by cold distillation, lysis the red blood cells in three steps, then digest the cell membrane and cell nucleus with lysis buffer containing non-ionic detergents, along with proteinase K, and then digest the cells. The use of phenol and chloroform digests proteins and introduces DNA into the aqueous phase. Eventually, the water molecules move away from the DNA around the alcohol and form a DNA coil. This extracted DNA is dissolved in water and measured by a nanodrop machine.
۲- RNA Extraction from Microorganisms, Blood, Cell Lines, Fresh Tissue in Molecular Genetics Laboratory:
Studying and analyzing gene expression changes under different experimental conditions, as well as under different physiological conditions, is one of the most important research areas in many molecular biology laboratories around the world. The study of gene expression is used in the study of cancer, stem cells, development and embryology, specific diseases and genetic conditions, infection rates of viruses such as HIV, HBV, etc.
In experiments related to the expression of genes, the macromolecule is the RNA studied. The Generators Lab provides RNA extraction services from blood, tissue, cell lines, bacteria, and plant tissues. RNA extraction services are performed with Trizol or with commercial kits from companies such as BIORAD, QIAGEN, ThermoFisher, and other leading brands in the world.
۳- Determination of the concentration and purity of nucleic acids by OD or NanoDrop reader:
The nanodrop machine is able to determine the concentration of nucleic acids in ng / µl in very small volumes, for example, 5 microliters. Also by determining the optical absorption ratio of nucleic acids (DNA, RNA) at 2 nm to optical absorption at 2 nm (protein adsorption) and optical absorption at 1 nm to optical absorption at 2 nm (optical absorption of organic matter such as phenol, respectively) Protein contamination and contamination of organic matter.
۴- cDNA synthesis:
One intermediate step in gene expression studies is cDNA synthesis. CDNA synthesis is performed by reverse transcriptase. In this process, the RNA extracted from the sample is converted to cDNA (complementary DNA). In this process, oligo (dt) or Random Hexamer primers are used. If oligo primers (dT) are used, only mRNAs are used as a template in the synthesis process. If the Random Hexamer primer is used, all the transcriptome content will be used as a template. Lab cDNA synthesis services are performed using commercial kits from the top brands currently available.
۵- Types of PCR such as ARMS-PCR, PCR-RFLP, RT-PCR:
Polymerase Chain Reaction (PCR) is the most widely used technique in molecular biology that is used to amplify one or more fragments of specific sequence DNA in large numbers. The technique was invented in the year 6 by Kerry Mullis. The basis of the PCR method is the enzymatic synthesis of a DNA sequence by DNA polymerase, which is carried out at multiple temperature cycles. These cycles consist of steps of double-stranded DNA cleavage, binding of primers to their target site on single-stranded DNA and then amplification. DNA is synthesized by the Taq polymerase enzyme and the construction of a new strand.
This technique can detect single nucleotide polymorphisms (SNPs) and mutations by ARMS-PCR, PCR-RFLP and tetra ARMS-PCR methods.
۶- Quantitative and absolute quantitative analysis of genes by Real-Time PCR and data analysis in molecular genetics laboratory:
Real-time PCR is also called qRT-PCR, one of the most sensitive tools used in molecular biology. The high sensitivity, widespread use, and reproducibility have made this method a special place worldwide in molecular biology laboratories.
Due to the addition of other SYBR Green in the real-time PCR reaction tube, it allows the observation of reaction progress online and in real time. But what makes real-time PCR so valuable is not just this. With this PCR method, it is possible to quantitatively analyze gene expression changes. By obtaining amplification plot and threshold setting, one can obtain the threshold cycle (Ct) for each sample. By obtaining Ct for the genes under study and a Ct for a reference gene (reference or housekeeping) as internal control, one can calculate Ct Delta (CtΔ) and then Delta Ct Delta (CtΔΔ). Finally, by calculating fold change, we can quantitatively report the expression changes of a gene in the studied samples. The Generators Lab offers a variety of real-time PCR, relative qRT-PCR and absolute qRT-PCR services using Cyber Green and TaqMan hydrolysis probes.
۷- High Resolution Melting (HRM) Genotyping Services:
The HRM technique is a powerful technique for detecting mutations and polymorphisms and epigenetic changes in DNA samples. It is also a fast and cost-effective technique that can be easily accomplished with a Real-time PCR, which has the advantage over TaqMan sequencing techniques. This method uses special fluorescent dyes, which do not have any radiation in the free state but emit fluorescent light when they are attached to the double stranded DNA. After PCR, they gradually warm to DNA to begin denaturation. As the DNA melts, the dye molecules break down, resulting in a decrease in fluorescence intensity. Fragments of the same length will have different melting temperatures even if they are different in one nucleotide
۸- Vertical electrophoretic (acrylic hope gel) and silver nitrate staining:
In electrophoresis, the DNA fragments move from the negative pole to the positive pole due to the negative charge of the phosphate group against the electric field and are separated by size. That is, the shorter the length of the piece, the longer the gel can travel and vice versa. Horizontal electrophoresis is usually performed on agarose gels and is visualized by UV bands after staining with ethidium bromide or substances such as Safe Stain.
PAGE (Polyacrylamide Gel Electrophoresis) is a very high resolution electrophoresis even up to a few bp that can be used for ARMS-PCR, PCR-RFLP and tetra ARMS-PCR methods for band separation and genotype detection. Omid Acrylic Gel contains Acid Omid powder and Bis Acryl Acid with TBE buffer to which TEMED and ammonium peroxisulfate catalysts are added to accelerate the polymerization reaction. For electrophoresis gel staining the samples were first fixed with alcohol and acetic acid and then stained with silver nitrate solution and finally visualized with the appearance of NaOH bands.
Vertical and horizontal electrophoresis services of this quality are offered in this laboratory.