Lab 6 Isolation Techniques

DNA Isolation from Haman Blood Cells Objective: Isolating DNA molecules from white blood cells (WBCs) and visualizing them directly. Blood is composed of liquid plasma and solid substances including blood cells. WBCs have nuclei that contains the nucleic acids. 1-Cellular membrane and nucleic membrane must be destroyed first 2- Other components of the cell like proteins must be excluded 3-finally DNA molecules can be collected and directly visualized.

Reagents : Chemicals such as In this experiment : 1- cellular and nucleic membranes are lysed with using lysis buffer 2- Proteins are precipitated by using phenol and chloroform. 3- Protect DNA from lysis by DNase enzyme by using EDTA which inhibit DNase 4- Collect DNA molecules by adding both sodium acetate and isopropanol. Reagents : Chemicals such as 1-EDTA (Ethylene diamine tetra acetate) which removes Mg+2 ions that is essential for preserving the overall structure of the cell membrane. 2-SDS (Sodium dodecyl sulfate), which aids in disrupting the cell membranes by removing the lipids of the cell membranes, are included in the extraction. 3-buffer: which lysing the cells, the final step in the preparation of a cell extract is removal of insoluble cell debris

Purification of DNA from cell extract: In addition to DNA the cell extract will contain significant quantities of protein and RNA. A variety of procedures can be used to remove these contaminants, leaving the DNA in a pure form. Reagents Phenol: The standard way to de-proteinize a cell extract is to add phenol or a 1:1 mixture of phenol: chloroform. These organic solvents precipitate proteins but leave the nucleic acids in aqueous solutions. The aqueous solution of nucleic acid can be removed with a pipette. Ribonuclease enzyme: Ribonuclease enzyme remove RNA by degrade these molecules into ribonucleotide subunits.

Materials: - Lysis buffer - SE buffer Blood with anti-coagulant - Proteinase K (10mg/ml) - SDS reagent (contains detergent and EDTA) - Chloroform/ isoamyl alcohol 24:1 - Sodium acetate - Isopropanol - Micropipettes and tips - Refrigerated centrifuge and 15 ml Eppendorf tubes

Steps: 1- In Eppendorf tube add 2.5 ml of blood with 7.5 ml of lysis buffer, shake well, and leave it in ice for 5 min. 2- Put the sample in the centrifuge (with balancing tube of 10 ml water) at 1200 rpm, 4˚C for 10 min. 3- Discard supernatant. Add to the pellet 2.5 of lysis buffer and centrifuge again at same conditions (set balance tube ) 4- Discard supernatant. Add 1.25 ml SE buffer to pellet, centrifuge again at same conditions (set balance tube !) 5- Discard supernatant. Add 1.25 ml of SE buffer + 10μl proteinase K + 60 μl SDS. Shake gently for 7 min.

6- Add 1. 25 ml SE buffer + 2. 5 ml phenol 6- Add 1.25 ml SE buffer + 2.5 ml phenol. Shake well for 5 min then centrifuge again at 3000 rpm, 10 ˚C for 5 min. 7- Transfer supernatant to new tube. Add 2.5 ml Phenol/ chloroform/isoamy alcohol. Shake well for 5 min, then centrifuge Again at same conditions. 8- Transfer supernatant to new tube. Add 2.5 ml chloroform/isoamyl alcohol. Shake well for 5 min, then centrifuge again at same conditions. 9- Transfer supernatant to new tube. Add 75 μl 3M Sodium acetate + 2.5 ml isopropanol. Observe DNA fibers visually. vortex shaker

DNA Isolation from Blood Animation http://www.iupui.edu/~wellsctr/MMIA/isolating_dna/dna_isolation_rev.swf