4 Isolation
ml 25% SDS
DNA
Protocol
Protocol.
ml 10% SDS
concentration
20 mL, 10%
Cell Lysis
Add 2 mL
20 ml of E. coli suspension
Add 0.4 ml
10% SDS,
25 % SDS to
to 10 mL
5 mL E. coli
E.coli cell
cell
suspension, mix well.
suspension,
Incubate at 65 oC for 10 min.
mix well.
Protein Removal
Incubate at
Aqueous layer, contains nucleic
Aqueous
acid acidlayer, contains
nucleic acidacidAdd 1.3 mL 5 M
65oC for 10
NaPerchlorate, mix well
min.
Add () mL chloroform in the fume
hood and shake with a mechanical
Protein Removal
Add 1.3 mL 5 M NaPerchlorate, mix well wrist arm shaker for 20 mins at
Add 6.7 mL chloroform in the fume hood room temperature
and shake with a mechanical wrist arm
denatured
Chloroform
proteins
layer, contains denatured proteins
shaker Chloroform
for 20 min atlayer,
roomcontains
temperature
5 ml of E. coli suspension Cell Lysis
Removing Chloroform:
Removing Chloroform:
Centrifuge and take off the bottom
chloroform layer
Carefully layer on 2 volumes of icecold 100% ethanol to the aqueous
(top) layer.
Collect the
nucleic acid
which
precipitates at
the interface by
twirling a glass
rod.
Collect the
nucleic acid
which
precipitates at
the interface by
twirling a glass
rod.