Isolation of Plasmid, isolation of plasmidspptx

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1. Presented By: M. Awais Zafar (2017-ag-1449) M.Phil Scholar (Theriogenology) MED-707 ISOLATION OF PLASMID

2. What is Plasmid??? “ Plasmid is autonomously replicating, extra chromosomal DNA molecules, distinct from normal DNA molecules and non essential for cell survival under non selective conditions” Found in Bacteria and sometimes in Yeast Size ranges from 1KB to 400KB Copies ranges from single to hundreds in a cell. J.Lederberg studied the cell hereditary and suggested the term “Plasmid” for all extra chromosomal determinants.

3. Types of Bacterial Plasmid?? Based on Function, divided in to 5 categories. Fertility(F) Plasmids: Capable of Conjugation Resistance(R) Plasmids: Have Antibiotic Resistant Genes (R Factors) Col Plasmids: Codes for Colicines Proteins that kills other bacteria Degrative Plasmids: Enable digestion of unusual substances like toluene/Salicylic Acid Virulence Plasmid: Turns the Bacteria in to Pathogenic.

4. Development of Plasmid Vectors?? Plasmids serve as important tools in genetics and biochemistry labs, where they are commonly used to multiply or express particular genes. Plasmids used in genetic engineering are called “ vectors” Vectors are vehicles to transfer genes from one organism to another and typically contain a genetic marker conferring a phenotype .

5. Isolation of Plasmids?? Material Required in Lab : Beaker, Micro Pipette, Pipette Tips, Vials, TE buffer with RNAase A, Bacterial culture, Ethanol 70%, Vortex mixer, Centrifuge machine.

6. Method.. ?? Take 1500 μ l bacterial culture in micropipette and put it into vial, then centrifuge it at 5000 rpm for 5mints at 4ĉ. Take out tube then remove supernatant carefully without disturbing the pellets. Take lysis solution І (100 μ l) from ice box and mix with pellets then vortex it. Take lysis solution П (2000 μ l) from ice box put it in tube containing pellets and lysis solution І then mix it by inverting the vials (5-6 times)

7. Continue.. Put vial in ice box for 1mints Take lysis Solution Ш (150 μ l) from ice box and add in vial and mix by inverting and store vial in ice box for another 5mints Centrifuge it at 5000 rpm for 5mints at 4ĉ. Remove the supernatant carefully and transfer it to another vial Take 200 μ l ethanol 70% and add in vial containing supernatants and vortex it Centrifuge at 5000 rpm for 5mints at 4ĉ.

8. Continue.. Remove supernatant without disturbing pellets and then allow vial to stand at room temperature for 2mints Take ethanol 1000 μ l and add in vial then centrifuge at 5000 rpm for 5mints Remove supernatant and put vial in rack and let ethanol to evaporate for 5-10mints Take 50 μ l of T.E buffer with RNAase A and add in vial then vortex it for 2mint Detect plasmid by doing Agarose gel electrophoresis with suitable marker.

9. Video demonstration..