Lab 6Isolation 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.

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. 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