DNA Introduction describes the Introduction of DNA into a laboratory fixed slide

The file describes the Introduction of DNA into a laboratory fixed slide

1. The cell The basic unit of any living organism. It contains a complete copy of the organism's genome. Cells are of many different types (e.g. blood, skin, nerve cells, etc.), but all can be traced back to one special cell, the fertilized egg.

2. Eukaryotes vs. prokaryotes Prokaryotic cells: lack a distinct, membrane-bound nucleus. E.g. ?? Eukaryotic cells : distinct, membrane-bound nucleus. Larger and more complex in structure than prokaryotic cells. E.g. ???

3. Genetic Material Containing Organelles Nucleus: The cell nucleus​ is a membrane bound structure that contains the cell's hereditary information and controls the cell's growth and reproduction. It is the command center of a eukaryotic cell and is commonly the most prominent organelle in a cell. Mitochondria : Mitochondria are structures within cells that convert the energy from food into a form that cells can use. Although most DNA is packaged in chromosomes within the nucleus, mitochondria also have a small amount of their own DNA. This genetic material is known as mitochondrial DNA or mtDNA. Chloroplast: It contains its own DNA, which is called chloroplast DNA, abbreviated as cpDNA and also known as plastome . Present in Plants.

4. DNA Structure DNA consists of two molecules that are arranged into a ladder-like structure called a Double Helix. A molecule of DNA is made up of millions of tiny subunits called Nucleotides. Each nucleotide consists of: Phosphate group Pentose sugar Nitrogenous base

5. Nucleoside and Nucleotide

6. DNA is extracted from human Blood cells for a variety of reasons Forensics: Evolution: Medical: Bio-engineering or cloning General research purposes

7. Before DNA Extraction Take blood (5ml-10ml) via sterile syringe Pore blood in to anticoagulant tube or a falcon tube containing 200 micro liter 1M EDTA Store at -80 for 4,5 hours or -20 for 1 day to provide cold stress to RBC,s Thaw blood properly before extraction and mix gently

8. Reagents required Lysis buffer (10mM Tris HCL, 2mMEDTA , pH 8.0) TNE buffer ( Tris HCL 10 mM , EDTA 2mM, NaCl 400mM) 10 % SDS Proteinase-K solution 20mg/ml 6M NaCl Phenol- Choloroform - Isoamylalcohol (PCI) ( 25:24:1) Chilled Isopropanol 75 % Ethanol Low TE buffer (10mM Tris , 0.2mM EDTA)

9. Major Steps Cell lysis and purification of leukocytes Proteins and Lipid degradation DNA purification

10. DNA Extraction (1 st day) Before starting the DNA extraction liquefy or thaw the samples of blood and take 250 ul (micro liter)Blood from stock. Add up to 1 ml of lysis buffer in 1 ml blood containing Eppendorf. Centrifuge at 6000 rpm for 10 minutes ( All centrifugation in successive phases should be done at 8C)

11. Remove 250 ul supernatant . Breakdown the pellet made at lowermost by taping it gradually. Add lysis buffer up to 1 ml. Centrifuge at 6000 rpm for 10 minutes ( All centrifugation in successive phases should be done at 8C) Remove 500 ul supernatant . Breakdown the pellet made at lowermost by taping it gradually. Add lysis buffer up to 1 ml. Centrifuge at 6000 rpm for 10 minutes ( All centrifugation in successive phases should be done at 8C)

12. Remove 750 ul supernatant . Breakdown the pellet made at lowermost by taping it gradually. Add lysis buffer up to 1 ml. Centrifuge at 6000 rpm for 10 minutes ( All centrifugation in successive phases should be done at 8C) Remove 900 ul supernatant . Breakdown the pellet made at lowermost by taping it gradually. Add lysis buffer up to 1 ml. Centrifuge at 6000 rpm for 10 minutes ( All centrifugation in successive phases should be done at 8C)

13. Remove all supernatant . Breakdown the pellet made at lowermost by taping it gradually. Add lysis buffer up to 1 ml. Centrifuge at 6000 rpm for 10 minutes ( All centrifugation in successive phases should be done at 8C ) Remove the supernatant leaving pellet and re-suspend pellet in 150 ul TNE buffer for 250 micro liter initial blood volume. Add 20 ul 10 % SDS and 2ul Proteinase K. Samples were incubated overnight in 37dnnn C shakers

14. Day 2 nd Whole digestion of the pellet is crisscross after one night incubation. If the pellet is not fully digested then add additional Proteinase K according to the quantity of undigested pellet. Once more incubated at 37C for 2-3 hours or till the pellet is fully digested.

15. Inorganic DNA Extraction Method: 1 st day protocol is same in both methods : We preferred inorganic DNA Extraction protocol for fresh samples. The tubes should kept on snow and added 150 ul saturated NaCl (6 M ) for 250 ul initial blood volume . The falcon tubes were shaken energetically and place on ice for a second time for 10-15 minutes. Centrifuged at 6000 rpm for 10 minutes to settle down the pellet (salts and proteins). S upernatant poured in a labeled Eppendorf. Centrifuged at 6000 rpm for 10 minutes

16. Organic DNA Extraction Method by Phenol, Chloroform, Isoamylalcohol (PCI): For old samples we applied Organic DNA Extraction method (PCI). 1 st day protocol is same in both methods: Phenol : Chloroform : Isoamylalcohol 25 : 24 : 1 Add 150 ul of PCI for 250 ul initial blood volume. The falcon tubes were shaken energetically and place on room temperature for 15-20 minutes. centrifuged at 6000 rpm for 10 minutes to settle down the pellet (salts and proteins). The supernatant poured in a labeled Eppendorf. Centrifuged at 6000 rpm for 10 minutes

17. The same amounts of Isopropanol were added and overturned the tubes moderately until DNA was obvious. The tubes were leaved for 5 minutes on ice. Again Centrifuged the samples at 6000 rpm for 10 minutes The supernatant were thrown away carefully The DNA pellet was wash away with 150 ul of 70 % ethanol for 250 ul initial blood volume.

18. Centrifuged again at 6000 rpm for 10 minutes 70 percent ethanol was removed wisely leaving the pellet. The DNA pellet was air dried out in 37C air dryer. Added 40 ul low T.E ( Tris HCL 10mM, EDTA 0.2mM)/ or in Injection Water The tubes were placed in 37C shaking incubator overnight to melt the DNA. Covered bands of Para film round the tip of the tubes.

19. 3 rd day Keep the tubes in a shaky water bath 70C for an hour to deactivate nucleases The tubes were left at room temperature to be chilled Samples were Spin briefly 2ml autoclaved tubes were labeled by side and cap. Tubes were labeled including pedigree number individuals ID. DNA samples were Aliquoted in duplication and stored at -20C conferring to pedigree number in marked and numbered Cryoboxes . The samples were Stored at -80 C for long term storage