DNA Isolation | Online Biotech Notes

 

DNA ISOLATION

Process of purification of DNA from sample using a combination of physical and chemical methods. The first isolation of DNA was done in 1869 by Friedrich Miescher.

For genetic analysis, which is used for scientific, medical or forensic purposes.

1. Dna Isolation from Blood

DNA Isolation from Blood

Procedure

● Blood + Anticoagulant (EDTA, contains Heparin ).

● Centrifuged(3000-4000 rpm), plasma is discarded.

● RBC lysis buffer is added, only WBC’s are left.

● Extraction buffer(breaks fatty cell membrane) + SDS( denatures histones and other binding protein) + Proteinase K(denatures

protein).

● Placed overnight(18 hrs, 50 C).

● Saturated phenol is added.

● PCI is added, centrifuged.

● CI is added and centrifuged.

● Sodium acetate and chilled ethanol/isopropanol is added.

● TE is added.

2. Dna Isolation from leaves

Dna Isolation from leaves

Reagents Used -

➢ CTAB Method

● CTAB stands for cetyl trimethyl ammonium bromide.

➢ BME

➢ CI

➢ Isopropanol

➢ Ethanol

➢ TE

➢ Sodium acetate, absolute alcohol

Procedure

● Cut young leaves(2-5 gm)

● Grind it using liquid nitrogen

● Take this powder in 50 ml of centrifuge tubes

● Add 15-25 ml warm CTAB(extraction buffer)

● Stir it and add 400ul BME and PvP, stir it and keep in water bath for 1.5 to 2 hrs at 65 degree C

● Remove the tubes, add chilled CI(24:1)

● Centrifuge at 10,000 rpm for 15 mins

● Take aqueous phase, add 6-10 ml chilled isopropanol

● Keep tube at -20 C for half an hour

● Centrifuge it, DNA will pallet out, discard the aqueous phase

● Add 70% ethanol, remove ethanol and keep it overnight

● Add RNase (5-10ul)

● Keep in water bath at 37 C

● Add CI, centrifuge it

● Take supernatant, add sodium acetate and absolute alcohol

● Centrifuge it, take DNA pallet

● Add 70% ethanol washing, add 100ul TE