Procedure for Chromosomal DNA extraction of gram-negative bacteria

Chromosomal DNA Extraction Procedure for Gram-Negative Bacteria

Steps	Description
1.	Make about 20 ml of LB broth in a 50 ml test-tube for each isolate.
2.	Inoculate appropriate loopful of desired microorganism in the LB broth and incubate for 24-48 hrs, depending upon the turbidity appearance.
3.	Take 1.5ml of fresh culture (LB) broth in Eppendorf or microfuge tube with the help of pipette.
4.	Centrifuge it at 5000 rpm for 10 min.
5.	Repeat the same process for double harvest of cell mass by discarding the supernatant.
6.	Add 200 microliter of Lysis buffer I and 10 microliter of lysozyme (0.5mg/ml).
7.	Vortex the mixture.
8.	Incubate the Eppendorf tube containing mixture at 37°C for 30 minutes.
9.	Add 1/10th volume of 10% SDS (about 20 microliter).
10.	Incubate it at room temperature for 10-15 minutes.
11.	Add 1/100th volume of Proteinase K (10mg/ml) about 2-3 microliter.
12.	Incubate at 37°C for 30-40 minutes.
13.	Add equal volume of freshly prepared Phenol: Chloroform (1:1).
14.	Mix well up and down the mixture gently (vigorous shaking is not allowed) and leave for 10-15 minutes.
15.	Centrifuge at 8000 rpm for 10-12 minutes.
16.	Collect aqueous fraction in new microfuge tube/ Eppendorf tube.
17.	Add equal volume of 3M Sodium acetate (300-350 microliter).
18.	Leave it in the ice bath for 1 hr. (precipitation).
19.	Centrifuge it at 10000 rpm for 10-15 minutes.
20.	Collect the pellet.
21.	Wash with 70% ethanol (1 mL) and centrifuge it at 10000 rpm for 8-10 minutes.
22.	Finally, dissolve the pellet in 100 microliter TE buffer.
23.	Store at -20°C until use.