Horizontal Agarose Gel Electrophoresis

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Introduction to Molecular & Cell Biology (Bio210A)
Instructor: Elmar Schmid, Ph.D.
Materials & Procedure
- DNA Gel Purification Necessary Equipment:

Water purifying system (e.g. Millipore system)
 to retrieve pure water with double-distilled quality
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Table balance
Vortex
Heating block or water bath (temperature-adjusted to 50oC)
Micro-centrifuge (with rotor for 1.5 ml reaction tubes)
Adjustable-volume digital pipettes (e.g. Eppendorf, Finnipette)
Plastic racks for 1.5ml reaction tubes
Paper towels
Marker pen
Required Materials:
-
QIAquick Gel Extraction Kit 50 (Qiagen: Cat # 28704)
Double-distilled water (= ddH2O)
96 – 100% ethanol
100% isopropanol
(Optional) 3M sodium acetate solution, pH 5.0
Autoclaved/sterile PP reaction tubes (1.5 ml)
Autoclaved pipette tips (yellow, blue)
Procedure:
a. Weigh the agarose gel slice containing your PCR-amplified DNA fragment in
the colorless PP reaction tube on the table balance
- make sure that you tare out the weight of the empty, autoclaved reaction tube
(also see Section 2.4)
- make sure that the gel slice weighs less than 400mg!
- Write down the weight of your gel slice into the box below!
Net weight (gel slice):
= ________ mg
b. Pipette three volumes of Kit Buffer QG per one volume of gel slice ( 100mg ~
100μl); e.g. 600 μl Buffer QG to 200mg of gel slice
 for gels with an agarose content of more than 2% use 6 volumes
of Buffer QG per volume of gel
c. Close the reaction tube and incubate the gel slice at 50oC for 10 min until the
agarose gel slice has been completely melted
- flick or vortex the tube every 2 – 3 min
1
Introduction to Molecular & Cell Biology (Bio210A)
Instructor: Elmar Schmid, Ph.D.
d. After melting of the agarose piece in the tube, check that the color in the tube
remained yellow
- a color change to orange or violet indicates that the pH is to high (= alkaline)
which would impair the binding of DNA to the silica membrane
- if this is the case re-adjust the pH to <7.5 by adding 10 μl of 3M sodium acetate,
pH 5.0 to the mix to achieve a color change back to yellow
e. Pipette 1 gel volume of isopropanol to the sample and mix by vortexing, e.g.
200μl in case of a 200mg gel slice
- this is especially important if the DNA size is < 500bp (like most PCR
amplification products!) or > than 4kb
f. Insert a new QIAquick spin column in a new 2ml PP collection tube and
pipette the complete volume of your gel extraction sample into the spin
column (see Graphic below)
- make sure that the volume of your sample is less than 800 μl
- do NOT touch the silica membrane in your spin column while pipetting the
sample volume into it!
Assembly of the silica column/collection tube DNA isolation unit & Sample
loading
Lid
QIAquick
Spin Column
Max. 800 μl
of
Gel Extraction
Mix
Silica Gel
Membrane
Transfer &
Loading
Insert
2 ml PP
Collection
Tube
1
2
3
Graphic©E.Schmid/2003
2
Introduction to Molecular & Cell Biology (Bio210A)
Instructor: Elmar Schmid, Ph.D.
g. Place the spin column assembly plus loaded sample into the rotor of the
microcentrifuge
 if you have only one sample, make sure you counterbalance the
rotor by inserting a water-filled collection tube into the opposite
slot in the centrifuge rotor !!
 counterbalance tube should have the same weight than the
spin column-collection tube assembly
h. Centrifuge the sample for 1min at > 10,000 x g (= 13,000 rpm) in the
Eppendorf-type micro-centrifuge
i. Take out the sample(s), discard the flow-through in the collection tube (e.g.
sink or container) and place the spin column back in the same, emptied
collection tube
j. Pipette 750 μl of Buffer PE to the QIAquick spin column and centrifuge again
for 1 min at > 10,000 x g (= 13,000 rpm) in the Eppendorf-type micro-centrifuge
(= Washing step)
- make sure that ethanol (96 – 100%) has been added to Buffer PE before
use at this step!
- The label on the PE Buffer bottle will tell how much has to be added! (Use
a 10ml glass pipette for doing this)
k. Take the samples out of the rotor, discard the flow through and place the spin
column back in the same, emptied collection tube
l. Centrifuge the QIAquick column-collection tube assembly again for 1 min at >
10,000 x g (= 13,000 rpm) in the Eppendorf-type micro-centrifuge
- this step removes residual Buffer PE and ethanol from the silica membrane
m. Take the samples out of the rotor and place the QIAquick column on top of a
clean, autoclaved 1.5ml PP reaction tube
n. Pipette 50 μl of sterile, DNAse/RNAse-free ddH2O on top of the silica
membrane of the spin column (= DNA Elution step) and let stand for 1 min at
room temperature on your bench
- again, make sure NOT to touch the membrane during this step!
- alternatively, Buffer EB (10mM Tris, pH 8.5) or TE buffer can be used at his step
- pH of the elution solution should be between pH 7.0 – 8.5 to achieve optimum
DNA elution from the silica membrane!
o. Centrifuge the spin column-reaction tube assembly for 1min at > 10,000 x g (=
13,000 rpm) in the Eppendorf-type micro-centrifuge to elute the silica
membrane-bound DNA
p. Discard the spin column and store the eluated DNA in the 1.5ml reaction tube
at – 20oC in the freezer for later use (see Ligation & Cloning lab part)
- make sure that you label the tube with a water-resistant marker pen
- write down content, date and name initials
q. Tightly screw cap all used solvents and solutions and store the components
of the QIAquick Gel Extraction kit on the shelf
3
Introduction to Molecular & Cell Biology (Bio210A)
Instructor: Elmar Schmid, Ph.D.
r. Clean the bench and make sure you discard all the wastes appropriately
using the designated containers and waste bottles
4
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