CTAB Procedure

In the hood:

96 well dish with bacteria

titertech

microtubes

glass pipette

Remove colonies from each well using the titertech and place them into the cover

Pipette up and down to thoroughly mix the colonies

Aliquot 300 µl of the culture into a microtube; save about 4 or 5 tubes

In the lab:

Centrifuge the culture for about 2 minutes or until a pellet has formed

Remove the supernatant

Resuspend the pellet in 576 µl TE, 15 µl of 20% SDS and 3 µl of 20 mg/ml proteinase K

Incubate for 1 hour at 37 ℃

Add 166 µl of 3M NaCl and mix thoroughly

Add 80 µl of 10% CTAB in 0.7 M NaCl and thoroughly mix

Incubate for 10 minutes at 65 ℃

In the hood in the back:

Add an approximately equal volume of chloroform (700 µl)

In the lab:

Centrifuge for 5 minutes at room temperature

In the hood in the back:

Remove white interface (should be able to be done by using pipetter)

Transfer supernatant to another microtube

Discard remaining solution in chloroform waste receptacle

Add an equal amount of phenol/chloroform

In the lab:

Centrifuge for 5 minutes

In the hood in the back:

Transfer supernatant to a new microtube

Discard remaining solution in proper waste receptacle

In the lab:

Add 0.6 vol of isopropanol and gently rock back and forth until white precipitant forms

Centrifuge for 30 minutes

Remove supernatant and wash pellet with 70% ethanol

Centrifuge the tube for 5 minutes

Remove supernatant

Redissolve the pellet in 100 µl TE/10