Isolation of bacterial DNA with Gentra Puregene kit (modified protocol)

transfer culture to 1.5ml microfuge tube

centrifuge for 10min at 12000rpm to pellet cells

decant or aspirate off supernatant

add 1 ml sterile PBS to pellet and vortex briefly to resuspend cells

centrifuge for 10 min at 12000rpm

decant of supernatant

repeat steps 4-6 one more time

resuspend pellet in 50ul PBS buffer

add 2ul of MetaPolyzyme solution (5mg/ml) (Sigma#MAC4L)

incubate overnight at 35C

add 310ul lysis solution (Qiagen #158113) to each tube. Pipette up and down gently to

resuspend cells

heat samples to 80ºC for 5-10 minutes to complete the lysis

**stop step: lysed cells are stable at room temperature

add 1.2ul of 10mg/ml RNase A per tube

invert tube gently to mix

incubate at 37ºC for 30-60 minutes

cool sample to room temperature

add 105ul of protein precipitation solution (Qiagen #158123)

invert tube gently several times to mix

place on ice for 30 minutes

centrifuge at 12000rpm for 3 minutes.

(The precipitated protein should form a tight pellet).

transfer supernatant to a fresh sterile tube (by pipette)

add 1-2ul of glycogen (20mg/ml) (Thermofisher #R0561) to  the supernatant and mix it by pipetting up and down

add 400ul of 100% isopropanol

invert tube gently 50 times to mix

(optionally: incubate at RT for 30 min to improve DNA yield)

pellet at 13000rpm for 3 minutes

carefully decant off isopropanol (the pellet might be loose)

add 500ul of 70% ethanol (cold), and invert tube a few times to wash the DNA pellet

centrifuge at 13000rpm for 3 minutes

pour off ethanol carefully – pellet may be loose

air dry pellet for 15-30 minutes

rehydrate DNA in ~35-50ul of 10mM Tris buffer (pH7.5-8.0)

(adjust for large or smaller pellet)