DNA Isolation from Blood via Phenol–Chloroform Extraction Method DNA
EXPERIMENT TITLE: DNA Isolation from Blood via Phenol–Chloroform Extraction Method DNA
AIM : Our aim it is the revealing of the DNA molecule from organically intact cellular structures.
INTRODUCTION: Isolation of DNA is needed for genetic analysis, which is used for scientific, medical, or forensic purposes. Scientists use DNA in a number of applications, such as introduction of DNA into cells and animals or plants, or for diagnostic purposes.(1)
The three basic steps of DNA extraction are; 1) Lysis 2) Precipitation and 3) Purification.
Lysis :the cell and the nucleus are broken open to release the DNA inside and there are two ways to do this. Precipitation : When you complete the lysis step, the DNA has been freed from the nucleus, but it is now mixed with mashed up cell parts. Precipitation separates DNA from this cellular debris.
Purification: Now that DNA has been separated from the aqueous phase, it can be rinsed with alcohol to remove any remaining unwanted material and cellular debris. At this point the purified DNA is usually re-dissolved in water for easy handling and storage.(2) (3)
Debris: It is the collapse of membrane and membrane-bound organelles by centrifugation. / Rs: India ruble
MATERIALS : Eppendorf tubes -Micropipette -Centrifuge -Gloves –TRIS -HCl -SDS –EDTA- NaCl -Phenol/Chloroform/Isoamyl alcohol -Ethanol- Proteinase K -Blood Sample -MgCl2
METHOD : STEP 1- Cell Breakage:
1. 1 ml blood with anticoagulant is taken and 1 ml Tris-EDTA buffer in pH 7.6 and mixed well. Tube is centrifuged at 3500 rpm for 5 minutes.
2. Discard the supernatant. Cell debris is washed and added the same TE buffer, this step is repeated twice and then, discard the supernatant.
3. Add 1 ml of lysis buffer on the pellet and incubate at 56°C for 3-4 hours.
Lysis Buffer 0.5 M Tris HCl, 20 mM EDTA, 10 mM NaCl, 1% SDS, 0.5 mg/ml Proteinase K
STEP 2- DNA Extraction:
4. At the end of the incubation, 500 μl is added phenol, mixed gently and centrifuged at 12000 rpm for 4 minutes.
5. Take the supernatant and add 1 ml of P:C:I (25:24:1) (500μl P:480μl C:20μl Isoamyl alcohol), mix well and tube is centrifuged at 12000 rpm for 1 minutes.
6. Take the upper phase to a new eppendorf tube and add C:I (24:1) mixture and centrifuge at 6500 rpm for 5 minutes.
7. Aqueous phase is taken to a new tube and double volume of EtOH is added.
STEP 3- Resuspension of DNA
8. Mixture is inverted thoroughly and enabled to precipitate DNA.
9. Freeze at -20°C for 1-2 hours. Then, centrifuge at 12000 rpm for 10 minutes and supernatant is removed.
10. 70% EtOH is added into pellet and this washing process should be done twice. Mixture is centrifuged at 12000 rpm for 5 minutes.
11. The supernatant is discarded, and the pellet portion is allowed to dry. However, it should not contact with too much air. It is sufficient to leave the lid of eppendorf tubes open for 3-4 minutes.
12. Pellet is solubilized with TE buffer or distilled water.
