MB504 Viva Preparation 2023 for BIO203 BIO301 BIO302 ZOO505 by Sulman Ali

BIO203,BIO301,BIO302,ZOO505 · Viva preparation 2023 by · SULMAN ALI · BS-ZOOLOGY · 1= DNA Extraction:

DNA extraction is a method to purify DNA by using physical and/or chemical methods from a sample separating DNA from cell membranes, proteins, and other cellular components.

Why we do DNA Extraction: To DNA Profiling, to separate genetic material from the rest of the cell (proteins, RNA, cell membrane, etc.), to test a newborn for a genetic disease, analyze forensic evidence, or study a gene involved in cancer.

Application of this Method: To Studying the genetic causes of disease and for the development of diagnostics and drugs. It is also essential for carrying out forensic science, sequencing genomes, detecting bacteria and viruses in the environment and for determining paternity.

Name: · Blood · Sample · Lysis · Buffer · A1 Buffer SDS · 10% · Proteinase · K · Phenol chloroform · isoamyl alcohol · (PCM) · Iso- · propanol · Ethanol · 70% · Evaporate organic compounds:

Quantity: 200ul 800ul Function To collect DNA To break down the blood separate the RBCs. To resuspension of cell pellet for plasmid DNA extraction. Linearize the proteins into primary, secondary, tertiary structures. To break down these proteins structures. To remove salts Separate upper layer and precipitate the DNA. To wash-up the extracted DNA and to remove contaminants from it. To dry-up the DNA at least 4 to 7 hours.

Method: Add one by one buffers in 200ul blood sample in the micro centrifuge tube. Vortex it for 30 sec and centrifuge it in 15000 rpm for 3 mints. In the same way add all the buffers and reagents one by one with same method. In last wash up the DNA by 70% ethanol and leave it to dry it at least 4 to 7 hours.

2= Gel- Electrophoresis:

It is a laboratory technique used to separate DNA, RNA or protein molecules based on their size and electrical charge, the molecules to be separated are pushed by an electrical field through a gel that contains small pores.

Why we do GelElectrophoresis: To separation of DNA fragments for DNA fingerprinting to investigate crime scenes. To analyze results of polymerase chain reaction. To analyze genes associated with a particular illness.

Application of this Method: DNA profiling for taxonomy studies to distinguish different species. To separation of DNA fragments for DNA fingerprinting to investigate crime scenes. To analyze results of polymerase chain reaction. To analyze genes associated with a particular illness.

1 gram Agarose powder 1x TAE Buffer 500ml Ethidium bromide

Procedure: To make a gel, agarose powder is mixed with an electrophoresis buffer and heated to a high temperature until all of the agarose powder has melted. The molten gel is then poured into a gel casting tray and a “comb” is placed at one end to make wells for the sample to be pipetted into. Run the gel by connecting DC voltage(120) supply and in last view this gel under the UV transilluminator.

3= Nanodrop (Spectrophotometer):

The Nanodrop Spectrophotometer is used primarily to measure DNA, RNA or protein concentrations in the UV/visible light range. It can be used to measure absorbance spectra or single wavelengths of these macromolecules in solution.

230 to 280 nm wavelength Is to agjusted

1. 8 to 2 ratio Pure DNA

Below 1.7 Contamination of phonetic compounds. Higher to 2.2 Contaminations of proteins

Why we do this method: to check the purity of the extracted DNA or RNA and their quantities and the contaminants in them.

Applications of Nanodrop (Spectrophotometer): to check the purity of the extracted DNA or RNA and their quantities and the contaminants in them.

Procedure: Simply pipet a drop of your DNA, RNA, or protein sample—as little as 1.0–2.0 µL—on the pedestal and pull down the arm. No cuvettes or capillaries are needed and the results appear in seconds. And set the software as we like.

4= Primers Details:

PCR primers are short pieces of single-stranded DNA, usually around 20 nucleotides in length.

Function of primers: to find targeted gene or for locating muted gene.

Annealing · temperature of · primers

48 to 62 oc

Length

18 to 22 or 30 There may be deference appears in +-2

GC Contents

Almost 60%