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 (% w/v) separeted optimally (kb) Low % 0.3 0.6 0.8 1.0 5.0-60 1.0-20 0.8-10 0.4-8 compress low MW bands, improve big fragments resolution 1.2 1.5 2.0 0.3-7 0.2-4 0.1-3 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 Dimethyl sulfoxide (DMSO) Ethidium bromide Formamide Sodium hydroxide Nitrile,Pvc, (Viton) Neoprene, Teflon Nitrile Natural rubber Natural rubber, Neoprene, Nitrile, Polyethilene, PVC, Teflon (Viton)
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!