Download Agarose Gel Electrophoresis: Best Practices

AGAROSE GEL
ELECTROPHORESIS:
BEST PRACTICES
(BACK TO THE BASICS)
Unit of Tropical Laboratory Medicine
April 2009
Marcella Mori
WORKFLOW OF AGAROSE GEL
ELECTROPHORESIS: THREE STEPS
“Agarose gel electrophoresis makes use of agarose slab
and electric field to separate linear nucleic acid polymers
(DNA/RNA) according to their molecular size”
Optimimal separation of the fragments is influenced by best
practices followed upon the three steps of the workflow in
agarose gel electrophoresis:
1. Gel preparation
2. Electrophoresis
3. Nucleic acid visualization
WORKFLOW OF AGAROSE GEL
ELECTROPHORESIS: GEL PREPARATION
Preparation of
electrophoresis (E)
buffer
Add the needed amount of powdered agarose to
the E buffer in an Erlenmeyer flask
WORKFLOW OF AGAROSE GEL
ELECTROPHORESIS: GEL PREPARATION
Heat the agarose solution
in a microwave oven to
allow agarose to dissolve
Prepare
the
frame,
position the comb and
pour the agarose in it
WORKFLOW OF AGAROSE GEL
ELECTROPHORESIS: ELECTROPHORESIS
Prepare the samples
in loading buffer,
load the samples
and
run
the
electrophoresis
Running of electrophoresis
WORKFLOW OF AGAROSE GEL ELECTROPHORESIS:
NUCLEIC ACID VISUALIZATION
Acquisition of gel picture under UV light and
analysis of the DNA/RNA fragments
0. BEFORE STARTING
•Identify the presence of the material and
equipment to be used
•Confirm all instruments and equipment are
working properly
•Check
reagents
for
precipitation
discoloration, cloudiness, and expiration
date
1. GEL PREPARATION: AGAROSE
CONCENTRATION
Which sample is going to be tested?
gDNA/ RNA
Digested gDNA (large and small fragments)
Plasmid DNA (vector+insert)
YAC (large fragments)
PCR products (<3kb)
What’s the size of the DNA fragment of
interest?
► Define the percentage of the agarose gel
1. GEL PREPARATION: AGAROSE
CONCENTRATION
Concentration of agarose Range of sizes that are
separeted optimally (kb)
(% w/v)
0.3
5.0-60
0.6
1.0-20
0.8
0.8-10
1.0
0.4-8
1.2
0.3-7
1.5
0.2-4
2.0
0.1-3
Low %
compress
low MW
bands,
improve
big
fragments
resolution
High %
compress
high MW
bands,
improve
small
fragments
resolution
1. GEL PREPARATION: BUFFERS
Electrophoresis is performed at neutral pH to avoid
changes in charge of DNA.
Two buffers are generally used in the labs (for which
sympathizers are found for one another):
1. TBE (Tris/Borate/EDTA)
2. TEA (Tris/Acetate/EDTA)
Resolving power is the same
TEA easily exhausted (to be used not more than once)
EDTA helps to prevent DNAase-RNAase activity
►
GEL IN WATER OR GEL IN 10X STOCK SOLUTION
1. GEL PREPARATION
Preparation of
electrophoresis (E)
buffer
Add the needed amount of powdered agarose to
the E buffer in an Erlenmeyer flask
1. GEL PREPARATION: CASTING THE GEL
•Prepare the casting tray (tape or appropriate frame)
•Make sure tray is clean, dry and there is no residual
detergent or other debris (gels will have spots in the
background and it may interfere with further analysis of gel)
•Level casting tray before gel is poured
•Do not disturb gel after it is poured and while
still liquid
•Remove bubbles with clean tip immediately
1. GEL PREPARATION: PROBLEMS IN GELS
•Measure weights and volumes accurately
•Make accurate calculations
calculation of the volume: thickness 5-8 mm
•The gel sets before is poured
•Molten gel leaks from the casting tray
•Air bubbles form around the wells
•The gel is too thin or too thick
•The comb has touched the bottom of the casting
tray during setting so that the wells have holes in
the bottom
•The gel is made up in the wrong buffer
•The gel is not level
LEVEL THE GEL: PROBLEMS
Comb
+
Gel
Gel support tray
1. GEL PREPARATION
Heat the agarose solution
in a microwave oven to
allow agarose to dissolve
Asbestos
gloves
CASTING THE GEL: 60°C
Before addition of EtBr/pouring cool down at 60°C
•Avoid cracking of the casting tray
•Avoid thermodilatation of the casting tray (can touch the
bottom of the combs so that the wells will form with holes
at the bottom)
•Avoid EtBr carrying steam
EtBr conc 0,1-0,5 µg/ml
EtBr before/after?
ELECTROPHORESIS
•Once the gel is polymerized, put the gel in the tank, and
only now remove the combs (avoid gel collapsing)
•Check whether the system is working (wo samples)/Check
the volume of the well
•Heat samples (2-3min at 65°-70°C) facultative/mix with
loading buffer
•Loading is a skill to be acquired!!!!
2 hands!!!!
ELECTROPHORESIS
•Do not electrophoresis the gel backwards
•The rate of migration of nucleic acid is proportional to the
voltage applied:
Electric field (volt/cm)
•Increasing the volt, decrease the range of sizes to be
separated
Quick run 15 volts/cm
Good resolution 3-4 volts/cm
•Gel tanks have a maximum safe voltage written on a side
VISUALIZATION
•Remember to wear gloves (EtBr!!!)
•The transluminator is UV light (254nm or 302nm)
Wear complete facial protection (no googles!!)
Expose yourself not too long!! (wrist and forearms)
•Photographing (exposition time?)
TROUBLESHOOTING
NO BANDS SEEN ON GEL AFTER
ELECTROPHORESIS
Electrophoresis unit is contaminated
GEL WITH MISSING LANES
Degradation is seen in some lanes only, problems on the sample
LANES DO NOT RUN STRAIGHT
LANES DO NOT RUN STRAIGHT
•Gel was not level when poured
•Electrophoresis cell was not level
•Not enough buffer in electrophoresis chamber
•Buffer was not flowing evenly
•Temperature of buffer fluctuated during run
•Broken electrodes
•Electrophoresis chamber or power module
needs to be serviced
LANES DO NOT RUN STRAIGHT
Slight curvature of end lanes is common: avoid sample loading
WRINKLED OR CURVED BANDS
Gel agarose may be too hot when poured
SOME WORDS ON SAFETY
ETHIDIUM BROMIDE AND OTHERS
•We saw three moments important for safety
dissolving the agarose/visualization under UV/EtBr
EtBr is strongly mutagenic. Although there is not enough
evidence yet to classify EtBr as a human carcinogen or
teratogen, this material must be considered a possible
carcinogen and reproductive toxin.
EtBr is readily absorbed through the skin.
CHEMICAL RESISTANT GLOVES
List of glove material likely to provide adequate protection for more
than 4 hours
Acrylamide
Nitrile,Pvc, (Viton)
Dimethyl sulfoxide
(DMSO)
Ethidium bromide
Neoprene, Teflon
Formamide
Natural rubber
Sodium hydroxide
Natural rubber, Neoprene,
Nitrile, Polyethilene, PVC,
Teflon (Viton)
Nitrile
ETHIDIUM BROMIDE
Minimize the amount used
Work in a designated labeled area
Wear 2 pairs of long cuffed disposable gloves when
handling EtBr and a fastened laboratory coat which is
correctly fitted to cover exposed skin
Dispose of EtBr containing gels as hazardous chemical
waste
THANK YOU FOR YOUR ATTENTION!