Molecular & Cell Biology Laboratory Manual
Instructor: Elmar Schmid, Ph.D.
Procedure 2: DNA Isolation from bacteria using the Silcia Spin Column
Method
The so-called silica spin column method is a simple and fast DNA isolation technique
which is based on the strong interaction of the negatively charged DNA molecule with the
positive net charges of porous silica oxide (Si-O2) filter material (silica gel membrane)
Before you get started with this method following the procedure below, make sure that you
check whether you have all the necessary materials and reagents on your bench top and
mark them off in the check list below.
Necessary Equipment:
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Check Mark ( √ )
Micro-centrifuge w. rotor f. 2ml tubes
(e.g. Eppendorf)
Vortex
Water bath
Thermo-incubator (adjusted to 95oC)
Water bath sonicator
Thermometer
Adjustable-volume pipettes
Ear protectors
Styrofoam floaters
Paper towels
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Required Materials & Reagents:
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Double-distilled water (= ddH2O)
QIAamp DNA Mini Kit 50
(Qiagen: Cat #513040)
Proteinase K solution (2ml; 600mU/ml)
(Qiagen: Cat # 19131)
Fresh overnight bacterial culture (1ml/student)
 culture is either Fusobacterium nucleatum (Fn)
or Thermus thermophilus (Tt)
1.5ml microcentrifuge reaction tubes
(Sterile, PP, Eppendorf type)
Sterile pipet tips (yellow, blue)
Sterile pipet tips w. aerosol barrier (crystal)
100% Ethanol p.a. (ice-cold)
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Molecular & Cell Biology Laboratory Manual
Instructor: Elmar Schmid, Ph.D.
Procedure:
a. Pipet 1ml of the bacteria overnight culture into a sterile 1.5ml PPmicrocentrifuge tube and close cap
- your instructor will tell you which bacteria you will be using
- make sure that you label the tube with a permanent marker pen
b. Place the tube(s) into the rotor of the microcentrifuge and pellet the bacteria
by centrifuging (spin) them for 5min at 5000x”g” (= 7500 rpm)
 make sure that the rotor is counter-balanced at this step
 immediately stop the centrifuge if you hear some rattling and
counter-balance your tubes in the rotor!
c. Decant the culture medium in the supernatant into an appropriate biohazard
container for liquid wastes and observe the bacterial cell pellet at the bottom
of the tube
 the cell pellet should be small and firmly packed; if not, centrifuge again!
Protocol for gram-negative or cell wall-less bacteria
(e.g. E.coli, Enterobacter aerogenes or Sulfolobus solfataricus)
= Protocol A
d. Pipet 20 μl of the Proteinase K solution (kept on ice) to the cell pellet, mix by
vortexing and place tube in a little Styrofoam float
e. Transfer float with tube into a water bath and incubate at 56oC for approx.
30min until the tissue is completely lysed
 a good indicator for complete cell lysis is a change in light transmission which
leads to an opaque appearance of the cell suspension
 hold the tube against the light and observe this effect!
 vortex the tube occasionally during this incubation step!
f. Pipet 200 μl of Kit Buffer AL to the tube, mix thoroughly by pulse-vortexing
for 15 sec. and sonicate the cell lysate 3 times for 15 sec in a water bath
sonicator
 wear ear protector during this step!
 place the tube on ice for 30 sec. in between each sonication
pulse cycle to prevent over heating of your sample!
g. Briefly centrifuge the tube with your cell lysate in a microcentrifuge for 10 –
15 sec to remove drop from inside the lid
Continue the protocol with Step o. below
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Molecular & Cell Biology Laboratory Manual
Instructor: Elmar Schmid, Ph.D.
Protocol for gram-positive and bacteria with cell walls and capsules
(e.g. Clostridium perfringens )
= Protocol B
h. Pipet 180 μl of a 20mg/ml lysozyme solution in Lysis Buffer A (for recipe see
the materials section above) to the cell pellet and resuspend the cell pellet
with your pipet using a blue tip
 your instructor will demonstrate how you do this professionally!
i. Place the tube(s) in a little Styrofoam float, put it into a water bath and
incubate the tube(s) for 30min at 37oC
j. Take the tube(s) out of the water bath and pipet 20μl of the Proteinase K
solution (kept on ice) plus 200μl of Kit Buffer AL to the bacterial cells in the
tube(s)
 put the Proteinase K solution back on ice after you used it to
prevent inactivation of this enzyme due to decay
k. Vortex to mix the reagents in the tubes
l. Place the tube(s) in a little Styrofoam float, place in a water bath and incubate
for another 30min at 56oC
 vortex the tubes occasionally during this incubation step to
improve the disruption of the bacterial cells
m. Take the tube(s) out of the water bath and treat the bacterial cells in the
tube(s) with 3 short (10 sec.) pulses of sonic waves created with the help of a
water bath sonicator; place the tube(s) on ice for at least 30 sec. in between
each sonication cycle
 your instructor will show you how you do this!
 wear ear protectors during this step due to the high pitch sound
waves
n. Briefly spin the tube(s) for a couple of seconds in the micro-centrifuge to
remove evtl. droplets from the inside of the lid
Continue with Step o.
o. Pipet 200μl ethanol p.a. (96-100%) to the bacterial cell lysate in the tube(s) and
thoroughly mix by pulse-vortexing for 15 sec.
 a white, cloudy precipitate will form after ethanol addition
 it is important to apply this precipitate together with the other
solution to the silica spin column in step q.
p. After mixing, briefly centrifuge the 1.5 ml microcentrifuge tube(s) in a
microcentrifuge to remove droplets from the inside of the lid and place them
in a plastic rack on your bench
q. Next, place a new QIAamp silica spin column on top of a new 2ml PP reaction
tube as shown in Graphic 1 below and securely place it in a plastic rack for
upcoming procedures
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Molecular & Cell Biology Laboratory Manual
Instructor: Elmar Schmid, Ph.D.
Graphic 1: Assembly of the silica column/collection tube DNA isolation unit &
Sample Loading
Lid
QIAamp Mini
Spin Column
Blue
400 μl Cell
Lysate Mix
Silica Gel
Membrane
(after Step p)
Insert
Transfer &
Loading
2 ml PP
Collection
Tube
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Graphic©E.Schmid/2003
r. Carefully pipette the mixture including the whitish precipitate in your 1.5 ml
microcentrifuge tube(s) (step p) into the top chamber of the silica spin
column without wetting the rim and without touching the white silica gel
membrane at the bottom of the top chamber
 use a new, sterile blue tip for transferring the mix
s. Close the cap of the QIAamp slica spin column and centrifuge the whole
assembly (= spin column plus 2ml PP collection tube) at 6000 x g (8,000 rpm)
or maximum speed for 1 min
t. Place the QIAamp silica spin column in a new, clean 2ml PP collection tube;
discard the old collection including the flow through
u. Carefully open the silica spin column and pipette 500 μl of Kit Buffer AW1
without wetting the rim of the column; close the cap and centrifuge the whole
assembly (= spin column plus 2ml PP collection tube) at 6,000 x g (8,000 rpm)
for 1min in a microcentrifuge (= 1. Washing step)
 make sure that ethanol has been added to wash buffer AW1
at the appropriate amount (see vial label) prior to this step
v. Place the QIAamp silica spin column in a new, clean 2ml PP collection tube;
discard the old collection including the flow through
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Molecular & Cell Biology Laboratory Manual
Instructor: Elmar Schmid, Ph.D.
w. Carefully open the silica spin column and pipette 500 μl of Kit Buffer AW2
without wetting the rim of the column; close the cap and centrifuge the whole
assembly (= spin column plus 2ml PP collection tube) at maximum speed (=
20,000 x g; 14,000 rpm) for 3 min in a microcentrifuge (= 2. Washing step)
 make sure that ethanol has been added to wash buffer AW1
at the appropriate amount (see vial label) prior to this step
x. Place the QIAamp silica spin column in a new, clean 2ml PP collection tube
and discard the old collection including the flow through; centrifuge again at
maximum speed (= 14,000 rpm) for 1 min
 this step helps to get rid of any residual Buffer AW2 from the
silica membrane which might otherwise interfere with many
steps after your DNA isolation, e.g. PCR amplification, cloning, etc.
y. Place the QIAamp silica spin column in a new, sterile 1.5 ml PP
microcentrifuge tube; discard the old collection including the flow through;
carefully open the silica spin column and pipette 100 μl of Kit Buffer AE or
DNA-free distilled water (ddH2O) without wetting the rim of the column and
close the cap again (= DNA Elution step)
z. Incubate the assembly at room temperature for 1 min on your bench, then
centrifuge the whole assembly (= spin column plus 1.5 ml microcentrifuge
tube) at 6,000 x g (8,000 rpm) for 1min in a microcentrifuge to finally elute
your bacterial DNA from the silica gel membrane
 you may add another 100 μl of Kit Buffer AE or DNA-free distilled
water (ddH2O) to the spin column and repeat step z to elute more
DNA from your column
 in this lab you don’t have to, since we will have enough DNA eluted
to do the PCR experiment in an upcoming lab (see PCR lab)
Info:
- According to the manufacturer of the QIAamp DNA Mini Kit, between 10 – 30 μg of
DNA can be isolated from 25mg of tissue or cell suspension
- The protocol above also purifies RNA but in a much lesser amount than DNA
- The absorption ration A260/A280 of the purified DNA material is between 1.7 and 1.9
Discard the used spin column, close the cap of you 1.5 ml microcentrifuge tube
with your just isolated bacterial DNA and freeze this tube (with your name
initials, date and brief info regarding tube content) at – 20oC in the freezer
section of the lab fridge
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