Question

Explain, in great detail, the protocol of DNA isolation. In your explanation, mention which steps and/or materials are important in the process, and explain the significance of these steps. Following your explanation, describe where, in the protocol, can contamination occur and cause DNA isolation to fail.

Answer 1

As per the question, below is protocol for E.Coli plasmid  DNA isolation

1. Harvest 5 ml the overnight grown bacterial cells in a centrifuge tube.

2. add 100 micro liter solution A or suspension buffer (50 mM glucose, 10 mM EDTA pH 8, 25 mM Tris pH 8, 100 micro gram / ml RNase H) and dissolve the cells in it. and keep on ice.

3. Now add 200 microliter of lysis buffer (0,2 N NaOH, 1% SDS) and mix by inverting the tube. solution will become viscous and clear.

4. add 200 microliter of neutralization buffer (3M potassium Acetate) and mix by inversion of tube. a precipitate will be formed in tube.

5. centrifuge the tube at 10,000 rpm for 5 minute and take out the supernatant in a fresh sterile tube.

6.add chloroform IAA (isoamyl alcohol) and mix by inverting.

7. again collect the supernatant in afresh tube.  

8.Add 1 ml of chilled ethanol and keep in -20 degree for 1 hour to overnight.

9. after 1 hr centrifuge the tube at 10,000 rpm for 10 minutes and discard the supernatant as the pDNA will form pellet at the base of tube.

10 Now wash the tube with 70% ethanol and discard supernatant.

11. air dry the tube to remove traces of alcohol in air. alcohol hinder further reactions.

12. dissolve the pDNA in 20 microliter of sterile distilled water and check the quality of the DNA on 1% agarose gel containing Ethidium bromide.

the most critical steps are addition of lysis solution because longer exposure may cause loss to plasmid DNA causing nick or it may become single stranded. lessor exposure leads to genomic DNA contamination in preparation.

70% washing step is also critical as most of the time pellet may be lost at this step if researcher is not careful. so after adding the 70% alcohol, give a brief spin to keep the pellet in tact in position and then discard the alcohol.