TARGET FORMAT AND HYBRIDIZATION CONDITIONS
1 TARGET FORMAT AND HYBRIDIZATION CONDITIONS 2 END LABELING PROCEDURES 3 PHOTOBIOTIN LABELING 4 NICK TRANSLATION 5 RANDOM PRIMED LABELING 6 DESIGN AND EVALUATION OF 16S RRNA-TARGETED OLIGONUCLEOTIDE PROBES FOR FLUORESCENCE IN SITU HYBRIDIZATION 7 EVALUATION OF ALTERATIONS IN THE TUMOR SUPPRESSOR GENES INK4A AND INK4B IN HUMAN BLADDER TUMORS 8 THE ?-TUBULIN GENE REGION AS A MOLECULAR MARKER TO DISTINGUISH LEISHMANIA PARASITES 9 DETECTION OF MALIGNANT PLASMA CELLS IN THE BONE MARROW AND PERIPHERAL BLOOD OF PATIENTS WITH MULTIPLE MYELOMA 10 DETECTION OF CHLAMYDIA TRACHOMATIS BY DNA AMPLIFICATION 11 ISOLATION AND IDENTIFICATION OF CAMPYLOBACTER SPECIES BY USE OF SELECTIVE ENRICHMENT, NUCLEIC ACID AMPLIFICATION METHODS, AND GENE PROBES 12 DIAGNOSIS OF HUMAN PAPILLOMAVIRUS USING IN SITU HYBRIDIZATION AND IN SITU POLYMERASE CHAIN REACTION CONTENTS 13 QUANTITATION OF IN SITU HYBRIDIZATION USING IMAGE ANALYSIS OF RADIOACTIVELY LABELED RNA PROBES 14 STABLE ISOTOPE PROBING FOR DETECTION OF METHANOTROPHS AFTER ENRICHMENT WITH 13CH4 15 DETECTION OF ENTEROCOCCI IN FRESHWATER AND SEAWATER (16S AND 23S RRNA ENTEROCOCCUS OLIGONUCLEOTIDE PROBES) 16 ANALYSIS OF 16S–23S RRNA INTERGENIC SPACER OF VIBRIO CHOLERAE AND VIBRIO MIMICUS FOR DETECTION OF THESE SPECIES 17 COLONY HYBRIDIZATION OF BACTERIAL ISOLATES WITH BURKHOLDERIA CEPACIA-SPECIFIC PROBES 18 IN SITU HYBRIDIZATION OF BURKHOLDERIA CEPACIA CELLS 19 DETECTION OF POLYCHLORINATED BIPHENYL-DEGRADING ORGANISMS IN SOIL 20 DETECTION OF SINGLE-COPY GENES IN DNA FROM TRANSGENIC PLANTS 21 DIFFERENTIATION BETWEEN TRANSCRIPTS OF GENES BELONGING TO SMALL FAMILIES DURING FRUIT RIPENING AND ABSCISSION 22 POLYMERASE CHAIN REACTION DETECTION OF INVASIVE SHIGELLA AND SALMONELLA ENTERICA IN FOOD 23 POLYMERASE CHAIN REACTION FOR DETECTION OF LISTERIA MONOCYTOGENES TARGET FORMAT AND HYBRIDIZATION CONDITIONS 1 SOUTHERN BLOTTING 1 1 INTRODUCTION THE ISOLATION OF SPECIFIC REGIONS WITHIN THE GENOME OF AN ORGANISM IS NOW NORMALLY ACCOMPLISHED BY POLYMERASE CHAIN REACTION (PCR) AMPLIFICATION USING PRIMERS SPECIFIC FOR THE REGION IN QUESTION HOWEVER, THERE ARE OCCASIONS WHERE THIS IS NOT POSSIBLE (LOSS OF PRIMER SITES RESULTING IN NO AMPLIFICATION OR IF THERE ARE NO PRIMERS AVAILABLE) IN THESE CASES THE DETECTION OF THE SEQUENCE OF INTEREST CAN BE ACHIEVED BY HYBRIDIZATION OF A LABELED PROBE TO RESTRICTED GENOMIC DNA IMMOBILIZED ON A MEMBRANE BY SOUTHERN BLOTTING (1) GENOMIC DNA IS FIRST DIGESTED BY ONE OR MORE RESTRICTION ENZYMES AND THE FRAGMENTS GENERATED SEPARATED BY GEL ELECTROPHORESIS THE AMOUNT OF DNA TO BE APPLIED TO THE GEL VARIES FROM APPLICATION TO APPLICATION IN GENERAL 10 µG OF HUMAN GENOMIC DNA IS NEEDED FOR THE DETECTION OF A SINGLE COPY GENE WHEN USING RADIOACTIVELY LABELED PROBES AND AN OVERNIGHT EXPOSURE TO X-RAY FILM THIS FIGURE CAN BE REDUCED IF THE TARGET IS EITHER A REPETITIVE ELEMENT (E G , RIBOSOMAL DNA) OR, IF PLASMID DNA OR PCR PRODUCTS ARE RUN ON THE GEL ONCE THE FRAG- MENTS ARE SEPARATED ON THE GEL THE DNA IS THEN DENATURED IN SITU AND TRANS- FERRED BY CAPILLARY TRANSFER TO EITHER A NITROCELLULOSE OR NYLON MEMBRANE THE DNA FRAGMENTS ARE THEN BOUND TO THE MEMBRANE, WHICH CAN THEN BE USED IN A HYBRIDIZATION REACTION 1 2 MATERIALS 1 2 1 SPECIFIC MATERIALS 1 3MM FILTER PAPER (WHATMAN), PAPER TOWELS, GLASS OR PLASTIC TRAY AND SUPPORT (A GEL CASTING TRAY TURNED UPSIDE DOWN), CLING FILM 2 HYBOND N MEMBRANE (AMERSHAM PHARMACIA BIOTECH) 3 DENATURING SOLUTION: 1 5 M NACL, 0 5 M NAOH 4 NEUTRALIZING SOLUTION: 1 5 M NACL, 0 5 M TRIS-HCL, PH 7 2, 0 001 M EDTA 5 20× SALINE SODIUM CITRATE (SSC): 3 M NACL, 0 3 M NA3 CITRATE 6 UV TRANSILLUMINATOR 7 100× DENHARDT’S SOLUTION: 10 G OF BOVINE SERUM ALBUMIN (BSA) FRACTION V, 10 G OF FICOLL 400, 10 G OF POLYVINYLPYRROLIDONE (PVP) IN 500 ML OF DISTILLED WATER STORE AT –20°C IN 10 ML ALIQUOTS 8 10% (W/V) SODIUM DODECYL SULFATE (SDS) 9 10 MG/ML SHEARED HERRING TESTIS DNA STORE AT –20°C 1 2 2 OPTIONAL MATERIALS 1 0 25 M HCL 2 0 4 M NAOH AND A SOLUTION OF 0 1× SSC, 0 1% (W/V) SDS, AND 0 2 M TRIS-HCL, PH 7 5 3 OVEN SET AT 80°C 4 VACUUM BLOTTING SYSTEM (VACUGENE SYSTEM FROM AMERSHAM PHARMACIA BIOTECH) 1 3 METHOD 1 3 1 PREPARATION OF THE GEL FOR TRANSFER 1 ELECTROPHORESE SAMPLES IN AN AGAROSE GEL (SEE NOTE 1) AND TRANSFER THE GEL TO A GLASS OR PLASTIC TRAY SLIGHTLY LARGER THAN THE GEL 2 IF THE FRAGMENTS FOR ANALYSIS ARE LARGE (>10 KB) THE EFFICIENCY OF TRANSFER CAN BE INCREASED BY DEPURINATING THE DNA ADD 0 25 M HCL TO THE TRAY CONTAINING THE GEL UNTIL THE GEL IS JUST COVERED (SEE NOTE 2) PLACE ON A ROCKING PLATFORM OR ORBITAL SHAKER AND AGITATE GENTLY FOR 15–25 MIN AT ROOM TEMPERATURE 3 REMOVE THE 0 25 M HCL, RINSE THE GEL WITH DISTILLED WATER AND COVER WITH DENA- TURING SOLUTION RETURN THE TRAY TO THE ROCKER AND SHAKE FOR 30 MIN AT ROOM TEM- PERATURE (OR 15 MIN AFTER THE DYE HAS RETURNED TO BLUE) 4 REMOVE THE DENATURING SOLUTION, RINSE THE GEL WITH DISTILLED WATER AND COVER WITH NEUTRALIZING SOLUTION SHAKE FOR 15 MIN AT ROOM TEMPERATURE 5 REPEAT WITH FRESH NEUTRALIZING SOLUTION 6 SET UP THE CAPILLARY BLOT 1 3 2 SETTING UP THE CAPILLARY BLOT 1 HALF FILL A GLASS OR PLASTIC TRAY OF A SUITABLE SIZE WITH 20× SSC (SEE NOTE 3) PLACE A SUPPORT IN THE TRAY (THE UPTURNED CASTING TRAY IN WHICH THE GEL WAS CAST) COVER THE SUPPORT WITH A WICK MADE FROM THREE SHEETS OF 3MM PAPER ALLOW THE 20× SSC TO WET THE WICK AND ENSURE THERE ARE NO AIR BUBBLES TRAPPED BETWEEN THE SHEETS AND THE SUPPORT (SEE NOTE 4) 2 CAREFULLY PLACE THE TREATED GEL ON THE WICK ENSURING THERE ARE NO AIR BUBBLES TARGET FORMAT AND HYBRIDIZATION CONDITIONS BETWEEN THE WICK AND THE GEL SURROUND THE GEL WITH CLING FILM (FIG 1) TO ENSURE THAT TRANSFER OCCURS THROUGH THE GEL AND NOT AROUND THE SIDES 3 PLACE THE MEMBRANE ONTO THE GEL (SEE NOTE 5) 4 WET TWO SHEETS OF 3MM PAPER CUT SLIGHTLY LARGER THAN THE MEMBRANE WITH 2× SSC AND PLACE THESE (ONE AT A TIME) ON TOP OF THE MEMBRANE ENSURING THERE ARE NO BUBBLES 5 PLACE A DRY SHEET OF 3MM PAPER ON TOP OF THE WET ONES REPEAT WITH ANOTHER SHEET OF 3MM PAPER 6 PLACE A STACK OF PAPER TOWELS 5–10 CM HIGH ON TOP OF THE 3MM PAPER AND COVER WITH A GLASS PLATE PUT A 500-G WEIGHT ON TOP OF THE GLASS PLATE 7 ALLOW TRANSFER TO PROCEED FOR SEVERAL HOURS (PREFERABLY OVERNIGHT) 8 AFTER BLOTTING CAREFULLY DISMANTLE THE STACK OF PAPER TOWELS, 3MM SHEETS, ETC TO EXPOSE THE MEMBRANE BEFORE REMOVING THE MEMBRANE MARK THE EDGES OF THE GEL WITH A PENCIL (IF DESIRED THE WELLS CAN ALSO BE MARKED) 9 REMOVE THE MEMBRANE AND RINSE CAREFULLY IN 2× SSC TO REMOVE ANY ADHERING PIECES OF AGAROSE 10 AIR-DRY THE MEMBRANE ON A SHEET OF 3MM PAPER 11 FIX THE DNA TO THE MEMBRANE EITHER BY BAKING AT 80°C FOR 2 H OR BY WRAPPING THE MEMBRANE IN CLING FILM AND PLACING DNA SIDE DOWN ON A UV TRANSILLUMINATOR FOR 2–5 MIN (SEE NOTE 6) 1 4 OPTIONAL METHODS FOR DNA TRANSFER 1 4 1 BIDIRECTIONAL TRANSFER TO TWO MEMBRANES IF REQUIRED THE DNA IN A GEL CAN SIMULTANEOUSLY BE TRANSFERRED TO TWO MEM- BRANES USING THE METHOD OF SMITH AND SUMMER (2) THIS METHOD IS OF BENEFIT IF MANY PROBES NEED TO BE HYBRIDIZED TO THE DNA IN A SHORT SPACE OF TIME FIGURE 1 SCHEMATIC OF A CAPILLARY SOUTHERN BLOT TARGET FORMAT AND HYBRIDIZATION CONDITIONS 1 PREPARE THE GEL FOR TRANSFER AS OUTLINED IN SUBHEADING 1 3 2 AFTER THE FINAL NEUTRALIZATION STEP COVER THE GEL IN 10× SSC AND SHAKE FOR 30 MIN 3 WET TWO SHEETS OF 3MM PAPER IN 2× SSC PLACE ONE ON A FLAT CLEAN SURFACE 4 PLACE A NYLON MEMBRANE ON TOP OF THE SHEET OF 3MM PAPER, ENSURING THERE ARE NO AIR BUBBLES BETWEEN THE 3MM AND THE MEMBRANE 5 CAREFULLY PLACE THE GEL ON TOP OF THE MEMBRANE DO NOT MOVE THE GEL ONCE IT IS IN CONTACT WITH THE MEMBRANE AS TRANSFER WILL START IMMEDIATELY 6 PLACE THE SECOND MEMBRANE ON TOP OF THE GEL FOLLOWED BY THE SECOND WET SHEET OF 3MM PAPER, AGAIN MAKING SURE THERE ARE NO AIR BUBBLES 7 PICK UP THE “GEL SANDWICH” AND PLACE ONTO A STACK OF PAPER TOWELS COVER THE TOP WITH A SIMILAR STACK OF TOWELS PLACE A SHEET OF GLASS ON THE TOP AND WEIGH DOWN AS BEFORE 8 ALLOW TRANSFER TO PROCEED AS BEFORE 1 4 2 VACUUM BLOTTING THERE ARE A NUMBER OF ALTERNATIVE METHODS FOR DNA TRANSFER FROM AGAROSE GELS TO MEMBRANES ONE OF THE BEST OF THESE IN TERMS OF SIMPLICITY AND SPEED IS VACUUM BLOTTING HERE THE DNA IS LITERALLY SUCKED OUT OF THE GEL ONTO THE MEM- BRANE AND THE ENTIRE PROCESS CAN BE CARRIED OUT BETWEEN 20 AND 60 MIN USING A VACUUM BLOTTING SYSTEM SEVERAL GELS CAN BE BLOTTED IN A SINGLE DAY THE SYSTEM USED IN OUR LABORATORY IS THE VACUGENE XL AVAILABLE FROM AMERSHAM PHARMACIA BIOTECH 1 SET UP THE VACUUM BLOT APPARATUS ENSURING THERE IS A LIQUID TRAP BETWEEN THE PUMP AND THE BLOTTER 2 PREWET THE SUPPORT SCREEN WITH DISTILLED WATER AND PLACE SHINY SIDE UP IN THE BLOTTER 3 PLACE A PLASTIC MASK ON THE SUPPORT SCREEN WITH A PRECUT HOLE SLIGHTLY SMALLER THAN THE GEL TO BE BLOTTED 4 POSITION THE MEMBRANE OVER THE HOLE IN THE MASK, ENSURING THERE ARE NO AIR BUBBLES BETWEEN THE MEMBRANE AND THE SUPPORT SCREEN 5 CAREFULLY PLACE THE GEL ON TOP OF THE MEMBRANE, ENSURING THAT THERE ARE NO AIR BUBBLES BETWEEN THE GEL AND THE MEMBRANE AND THAT THE EDGES OF THE GEL PROTRUDE OVER THE HOLE IN THE PLASTIC MASK 6 CLAMP THE TOP OF THE BLOTTING APPARATUS TO THE LOWER PART CONTAINING THE GEL 7 SWITCH ON THE VACUUM PUMP AND POUR 0 25 M HCL INTO THE APPARATUS SO THAT IT COVERS THE GEL STABILIZE THE VACUUM AT 50 MBAR AND LEAVE FOR 4 MIN 8 REMOVE THE 0 25 M HCL BY TILTING THE APPARATUS AND SUCKING OFF THE SOLUTION THIS CAN BE ACHIEVED BY HAVING A “T” CONNECTOR BETWEEN THE LIQUID TRAP AND THE BLOTTER, WHICH CAN BE OPENED AND CLOSED BY MEANS OF A CLIP RESIDUAL SOLUTIONS CAN BE REMOVED FROM THE GEL SURFACE BY WIPING WITH A GLOVED FINGER OR A DISPOS- ABLE PIPET 9 POUR IN THE DENATURING SOLUTION UNTIL IT COVERS THE GEL AND LEAVE FOR 3 MIN REMOVE AS BEFORE TARGET FORMAT AND HYBRIDIZATION CONDITIONS 10 COVER THE GEL WITH NEUTRALIZING SOLUTION (1 0 M TRIS-HCL, PH 5 0, 1 5 M NACL, 0 001 M EDTA), LEAVE FOR 3 MIN, AND REMOVE 11 COVER THE GEL WITH 20× SSC AND LEAVE FOR 20–60 MIN (SEE NOTE 7) MAKE SURE THE GEL REMAINS IMMERSED DURING TRANSFER 12 REMOVE THE 20× SSC AND WITH THE VACUUM STILL APPLIED PEEL THE GEL OFF THE MEM- BRANE SWITCH OFF THE VACUUM AND REMOVE THE FILTER TREAT AS BEFORE 1 5 HYBRIDIZATION CONDITIONS THERE ARE MANY DIFFERENT HYBRIDIZATION SOLUTIONS IN THE LITERATURE THE ONE DETAILED HERE IS SIMPLE TO MAKE AND GIVES LOW BACKGROUND WITH THE HYBOND N NYLON MEMBRANES THE HYBRIDIZATION CAN BE CARRIED OUT IN EITHER HEAT SEALED PLASTIC BAGS THAT CAN WITHSTAND THE NECESSARY TEMPERATURES OR IN PLASTIC BOXES WITH SEALABLE LIDS 1 MAKE UP A PREHYBIDIZATION SOLUTION THAT CONTAINS FINAL CONCENTRATIONS OF 5× SSC, 5× DENHARDT’S SOLUTION, AND 0 5% SDS ALLOW 125 µL OF SOLUTION PER CM2 OF MEMBRANE PLACE THE PREHYBRIDIZATION SOLUTION IN A 50-ML TUBE AND PLACE IN A 65°C WATER BATH 2 BOIL ENOUGH HERRING TESTIS DNA TO GIVE A FINAL CONCENTRATION OF 100 µG/ML FOR MIN AND SNAP COOL ON ICE ADD TO THE PREHYBRIDIZATION SOLUTION 3 PREWET THE MEMBRANE TO BE HYBRIDIZED IN 5× SSC AND PLACE IN AN OPENED OUT PLASTIC BAG (SEE NOTE 8) CLOSE THE BAG OVER THE FILTER AND HEAT SEAL AROUND THE EDGES AS CLOSE TO THE GEL AS POSSIBLE LEAVING THE TOP OPEN 4 POUR IN THE PREHYBRIDIZATION SOLUTION, SQUEEZE OUT AS MUCH AIR AS POSSIBLE, AND SEAL THE TOP OF THE BAG WITH A HEAT SEALER 5 PLACE THE BAG BETWEEN TWO SHEETS OF GLASS AND PLACE IN A SHAKING 65°C WATER BATH INCUBATE FOR AT LEAST 30 MIN 6 DENATURE THE LABELED PROBE BY BOILING FOR 5 MIN AND SNAP COOLING ON ICE CUT ONE CORNER OFF THE HYBRIDIZATION BAG AND PIPET THE PROBE INTO THE PREHYBRIDIZATION SOLUTION RESEAL THE BAG AND INCUBATE AT 65°C IN A SHAKING WATER BATH OVERNIGHT 7 PREPARE WASH SOLUTIONS (1–5 ML/CM2 MEMBRANE) AND PREHEAT TO 65°C 8 AT THE END OF THE HYBRIDIZATION CAREFULLY CUT ONE CORNER OFF THE BAG AND POUR THE HYBRIDIZATION SOLUTION INTO SUITABLE CONTAINER FOR DISPOSAL OPEN THE BAG AND REMOVE THE MEMBRANE AND PLACE IN A SEALABLE PLASTIC BOX 9 WASH THE MEMBRANE BY INCUBATING AT 65°C IN A SHAKING WATER BATH IN THE FOLLOW- ING SOLUTIONS: 2× SSC, 0 1% SDS FOR 5 MIN (REPEAT), 1× SSC, 0 1% SDS FOR MIN, 0 1× SSC, 0 1% SDS FOR 10 MIN (REPEAT) (SEE NOTE 9) 10 REMOVE THE MEMBRANE FROM THE LAST WASH SOLUTION AND DRAIN THE EXCESS LIQUID OFF WRAP IN CLING FILM AND EXPOSE TO X-RAY FILM (SEE NOTE 10) IF THE MEMBRANE IS TO BE REPROBED IT MUST BE KEPT MOIST 1 6 PROBE REMOVAL FROM NYLON MEMBRANES IF THE MEMBRANE NEEDS TO BE HYBRIDIZED WITH MORE THAN ONE PROBE THE OLD PROBE CAN BE REMOVED FROM NYLON MEMBRANES (PROVIDING THEY HAVE NOT DRIED OUT) USING THE FOLLOWING PROCEDURE TARGET FORMAT AND HYBRIDIZATION CONDITIONS 1 PLACE THE MEMBRANE IN A SEALABLE PLASTIC BOX COVER THE MEMBRANE WITH 0 4 M NAOH AND INCUBATE AT 45°C FOR 30 MINS IN A SHAKING WATER BATH 2 POUR OFF THE NAOH SOLUTION AND COVER THE MEMBRANE WITH 0 1× SSC, 0 1% SDS, 0 2 M TRIS-HCL, PH 7 5 INCUBATE AT 45°C FOR 15 MIN IN A SHAKING WATER BATH 3 WRAP THE MEMBRANE IN CLING FILM AND EXPOSE TO X-RAY FILM TO ENSURE THE OLD PROBE HAS BEEN REMOVED 4 FILTERS CAN BE STORED WRAPPED IN CLING FILM AT –20°C INDEFINITELY 1 7 NOTES 1 FOR GENOMIC DNA TRANSFER USE AGAROSE (TYPE I; LOW EEO FROM SIGMA) AS THIS ALLOWS GOOD TRANSFER OF THE DNA OUT OF THE GEL AND IS FAIRLY CHEAP 2 WHEN THE SAMPLES ARE LOADED ON THE GEL USE A LOADING BUFFER CONTAINING 0 25% (W/V) BROMOPHENOL BLUE THE DEPURINATION STEP CAN BE MONITORED BY THE CHANGE IN COLOR OF THE DYE FROM BLUE TO YELLOW ONCE THE DYE HAS CHANGED COLOR LEAVE THE GEL FOR AN ADDITIONAL 10 MIN 3 AN ALTERNATIVE TRANSFER BUFFER IS 20× SSPE: 3 6 M NACL, 0 2 M SODIUM PHOSPHATE, 0 02 M EDTA, PH 7 7 HOWEVER, 20× SSC IS CHEAPER 4 AIR BUBBLES ARE EASILY REMOVED BY ROLLING A DISPOSABLE PIPET GENTLY OVER THE SUR- FACE THIS METHOD CAN BE USED AT ALL STAGES OF SET UP 5 SMALL DNA FRAGMENTS WILL START TO TRANSFER TO THE MEMBRANE IMMEDIATELY ON CON- TACT THEREFORE, DO NOT MOVE THE MEMBRANE ONCE IT ESTABLISHES CONTACT WITH THE GEL NYLON MEMBRANES DO NOT REQUIRE PRE WETTING BEFORE APPLICATION TO THE GEL IF A NITROCELLULOSE MEMBRANE IS TO BE USED FLOAT THE MEMBRANE ON THE SURFACE OF A TRAY FILLED WITH DISTILLED WATER UNTIL IT IS COMPLETELY WET CAREFULLY IMMERSE THE MEMBRANE AND LEAVE FOR 5 MIN 6 THE OPTIMUM EXPOSURE TIME VARIES BETWEEN TRANSILLUMINATORS AND CAN ALSO CHANGE WITH THE AGE OF THE UV BULBS TO CALIBRATE THE TRANSILLUMINATOR RUN A GEL CONTAIN- ING SIX LANES WITH 50 PG OF ? DNA DIGESTED WITH HINDIII IN EACH LANE BLOT THE DNA ONTO A MEMBRANE AND CUT THE FILTER INTO SIX STRIPS EXPOSE EACH STRIP FOR VARYING LENGTHS OF TIME RANGING FROM 30 S TO 10 MIN HYBRIDIZE THESE BLOTS TO ? DNA AND EXPOSE TO X-RAY FILM THE OPTIMUM EXPOSURE TIME CAN BE DETERMINED BY THE STRIP WHICH GIVES THE STRONGEST SIGNAL 7 TRANSFER TIMES VARY DEPENDING ON THE THICKNESS AND CONCENTRATION OF THE GEL, THE SIZE OF THE FRAGMENTS TO BE TRANSFERRED, AND THE LEVEL OF VACUUM APPLIED 8 SEVERAL MEMBRANES CAN BE HYBRIDIZED IN THE SAME BAG WITH LITTLE LOSS OF SIGNAL 9 IF A RADIOACTIVE PROBE IS USED THE PROGRESS OF THE WASHES CAN BE MONITORED USING A HAND HELD COUNTER THE MEMBRANE IS READY FOR AUTORADIOGRAPHY WHEN THE COUNTS FALL TO NEAR BACKGROUND ON AREAS OF THE MEMBRANE CONTAINING NO DNA IF IN DOUBT STOP THE WASHES EARLY AND EXPOSE TO X-RAY FILM IT IS ALWAYS POSSIBLE TO WASH THE MEMBRANE FURTHER IF THE SIGNAL IS TOO STRONG 10 EXPOSURE TIME VARY DEPENDING ON THE AMOUNT OF DNA RUN ON THE GEL, THE SPECIFIC ACTIVITY AND NATURE OF THE PROBE A PROBE HYBRIDIZING TO A SINGLE COPY SEQUENCE WILL REQUIRE LONGER EXPOSURE TIME THAN ONE FOR A REPETITIVE ELEMENT THE OPTIMUM EXPOSURE TIME WILL NEED TO BE DETERMINED FOR EACH EXPERIMENT IN GENERAL, AN TARGET FORMAT AND HYBRIDIZATION CONDITIONS OVERNIGHT EXPOSURE SHOULD SUFFICE FOR MOST APPLICATIONS EXPOSURE TIMES CAN BE SHORTENED BY PREFLASHING THE X-RAY FILM TO DO THIS MOUNT A FLASH GUN ON A SUP- PORT ABOUT 50 CM ABOVE THE BENCH IN THE DARKROOM COVER THE LENS OF THE FLASH GUN WITH SEVERAL LAYERS OF PAPER TO REDUCE THE AMOUNT OF LIGHT EMITTED TAKE A PIECE OF X-RAY FILM AND PLACE BELOW THE FLASH GUN COVER FOUR FIFTHS OF THE FILM WITH A SHEET OF CARD AND FIRE THE FLASH GUN MOVE THE SHEET OF CARD SO THAT THREE FIFTHS OF THE FILM IS EXPOSED AND FIRE THE FLASH GUN AGAIN REPEAT UNTIL THE ENTIRE FILM IS EXPOSED DEVELOP THE FILM TO DETERMINE THE OPTIMUM FLASH TIME FOR THE FILM/FLASH GUN COMBINATION THE REQUIRED EXPOSURE DOES NOT ALTER THE BACKGROUND OF THE FILM WHEREAS THE NEXT EXPOSURE DOES FLASHING THE X-RAY FILM IN THIS WAY PRESENSITIZES THE FILM, THUS REDUCING EXPOSURE TIMES 2 SLOT/DOT BLOTS 2 1 INTRODUCTION IF LARGE NUMBERS OF SAMPLES NEED TO BY HYBRIDIZED TO A PROBE THAT YIELDS A POSITIVE/NEGATIVE RESULT (SUCH AS SPECIES SPECIFIC CLONES), THIS CAN BE ACHIEVED BY DOT OR SLOT BLOTTING USING THIS TECHNIQUE DNA IS APPLIED DIRECTLY TO A MEM- BRANE AND THEREFORE NO GEL ELECTROPHORESIS IS REQUIRED COMMERCIAL MANIFOLDS ARE AVAILABLE WHICH CAN BE ATTACHED TO A VACUUM SOURCE WHERE THE DNA IS APPLIED TO WELLS FROM WHICH IT IS SUCKED ONTO THE MEMBRANE IN AN ORDERED ARRAY ALTERNATIVELY, THE DNA CAN BE PIPETTED DIRECTLY ONTO THE MEMBRANE USING A MICROPIPET 2 2 MATERIALS 2 2 1 SPECIFIC MATERIALS 1 3MM FILTER PAPER (WHATMAN) 2 HYBOND N MEMBRANE (AMERSHAM PHARMACIA BIOTECH) 3 DENATURING SOLUTION: 1 5 M NACL, 0 5 M NAOH 4 NEUTRALIZING SOLUTION: 1 5 M NACL, 0 5 M TRIS-HCL, PH 7 2, 0 001 M EDTA 5 20× SSC: 3 M NACL, 0 3 M NA3 CITRATE 6 UV TRANSILLUMINATOR 2 2 2 OPTIONAL MATERIALS 1 COMMERCIAL DOT/SLOT BLOT APPARATUS AND VACUUM SOURCE (E G , MINIFOLD SLOTBLOTTER OR DOTBLOTTER BY SCHLEICHER & SCHUELL UK LTD) 2 3 METHOD 1 HEAT DNA SAMPLES TO 95°C AND SNAP-CHILL ON ICE ADD AN EQUAL VOLUME OF 20× SSC 2 PLACE A MEMBRANE ON TOP OF A SHEET OF 3MM PAPER 3 SPOT THE SAMPLES ONTO THE MEMBRANE PREWETTED IN 10× SSC IN 2-µL ALIQUOTS TARGET FORMAT AND HYBRIDIZATION CONDITIONS ALLOWING TO DRY BETWEEN APPLICATIONS TAKE CARE NOT TO ALLOW SAMPLE SPOTS TO MERGE INTO EACH OTHER IF A DOT BLOT APPARATUS IS USED TURN ON THE VACUUM SOURCE AND PIPET THE SAMPLES INTO THE WELLS OF THE APPARATUS 4 IMMERSE THE MEMBRANE IN DENATURING SOLUTION FOR 5 MIN 5 TRANSFER TO NEUTRALIZING SOLUTION FOR 1 MIN 6 DRY AND FIX THE DNA TO THE MEMBRANE AS FOR SOUTHERN BLOTS 7 HYBRIDIZE TO A LABELED PROBE AS FOR SOUTHERN BLOTS 3 COLONY BLOTS 3 1 INTRODUCTION ISOLATION OF SPECIFIC SEQUENCES FROM DNA LIBRARIES CLONED IN EITHER BACTE- RIOPHAGE OR PLASMIDS CAN BE ACHIEVED BY PLATING THE LIBRARY OUT ON AGAR PLATES AND TAKING COLONY LIFTS FROM THE AGAR PLATES THE MEMBRANES CAN THEN BY USED IN A HYBRIDIZATION REACTION USING A SUITABLE PROBE 3 2 MATERIALS 3 2 1 SPECIFIC MATERIALS 1 HYBOND N NYLON MEMBRANES (AMERSHAM PHARMACIA BIOTECH) OF THE DESIRED DIAMETER (SLIGHTLY LESS THE DIAMETER OF THE AGAR PLATES THE COLONIES ARE GROWN ON) 2 3MM FILTER PAPER (WHATMAN) 3 STERILE NEEDLE AND BLUNT-ENDED FORCEPS 4 DENATURING SOLUTION: 1 5 M NACL, 0 5 M NAOH 5 NEUTRALIZING SOLUTION: 1 5 M NACL, 0 5 M TRIS-HCL, PH 7 2, 0 001 M EDTA 6 20× SSC: 3 M NACL, 0 3 M NA3 CITRATE 7 UV TRANSILLUMINATOR 3 2 2 OPTIONAL MATERIALS 1 10% (W/V) SDS 2 OVEN SET AT 80°C 3 3 METHOD 1 PLATE OUT THE BACTERIAL CELLS OR BACTERIOPHAGE ON AGAR PLATES AND INCUBATE OVER- NIGHT (SEE NOTE 1) COOL TO 4°C FOR AT LEAST 30 MIN 2 BEND THE MEMBRANE INTO A U SHAPE PLACE THE BOTTOM OF THE U IN CONTACT WITH THE SURFACE OF THE AGAR PLATE AND GENTLY FOLD DOWN SO THE ENTIRE MEMBRANE IS IN CON- TACT WITH THE AGAR PLATE DO NOT MOVE THE FILTER ONCE IT IS IN CONTACT WITH THE SURFACE OF THE PLATE AS THIS WILL RESULT IN SMEARING 3 MARK THE ORIENTATION OF THE MEMBRANE WITH RESPECT TO THE PLATE BY MAKING THREE ASYMMETRIC HOLES WITH A STERILE NEEDLE THESE CAN BE USED FOR ORIENTATION OF THE FILTER AFTER HYBRIDIZATION 4 REMOVE THE MEMBRANE AFTER 30–60 S WITH A PAIR OF BLUNT-ENDED FORCEPS MIN 4 NORTHERN BLOTS 4 1 INTRODUCTION RNA MUST BE RUN ON AGAROSE GELS UNDER DENATURING CONDITIONS TWO COM- MON METHODS CAN BE USED TO ACHIEVE THIS THE GLYOXAL–DIMETHYL SULFOXIDE (DMSO) METHOD AND THE FORMALDEHYDE/FORMAMIDE METHOD THE LATTER METHOD IS SLIGHTLY EASIER AND IS DESCRIBED HERE WHEN WORKING WITH RNA ALL GLASSWARE SHOULD BE BAKED AT 180°C OVERNIGHT AND ALL SOLUTIONS MADE UP CONTAINING 0 2% (V/V) DIETHYLPYROCARBONATE (DEPC) AND THEN AUTOCLAVED TO REMOVE CONTAMI- NATING RNAASES IT SHOULD BE NOTED THAT TRIS SOLUTIONS CANNOT BE DEPC-TREATED AND THAT TRIS STOCK SOLUTIONS SHOULD BE MADE UP WITH DEPC TREATED WATER THE TRIS STOCK FOR RNA WORK SHOULD BE TAKEN FROM A SEPARATE CONTAINER FROM NOR- MAL LABORATORY STOCKS AND SHOULD BE WEIGHED OUT ONLY BY SHAKING THE TRIS OUT OF THE CONTAINER (NEVER USE A SPATULA) 4 2 SPECIFIC MATERIALS 1 DEPC FOR TREATING ALL SOLUTIONS TO BE USED CAUTION: DEPC IS A VERY DANGEROUS SUBSTANCE AND CARE MUST BE EXERCISED WHEN HANDLING IT ONCE AUTOCLAVED THERE IS NO FURTHER HAZARD 2 10× MOPS BUFFER: 0 2 M 3-[N-MORPHOLINO] PROPANESULFONIC ACID, 0 5 M NA ACETATE, PH 7 0; 0 01 M EDTA 3 FORMALDEHYDE: 37% SOLUTION, 12 3 M, PH >4 0 TARGET FORMAT AND HYBRIDIZATION CONDITIONS 4 FORMAMIDE (DEIONIZED) 5 3MM FILTER PAPER (WHATMAN), PAPER TOWELS, GLASS OR PLASTIC TRAY AND SUPPORT (A GEL CASTING TRAY TURNED UPSIDE DOWN), CLING FILM 6 HYBOND N MEMBRANE (AMERSHAM PHARMACIA BIOTECH) 7 DENATURING SOLUTION: 1 5 M NACL, 0 5 M NAOH 8 NEUTRALIZING SOLUTION: 1 5 M NACL, 0 5 M TRIS-HCL, PH 7 2, 0 001 M EDTA 9 20× SSC: 3 M NACL, 0 3 M NA3 CITRATE 10 UV TRANSILLUMINATOR 11 100× DENHARDT’S SOLUTION: 10 G OF BSA FRACTION V, 10 G OF FICOLL 400, 10 G OF PVP IN 500 ML OF DISTILLED WATER STORE AT –20°C IN 10 ML ALIQUOTS 12 10% (W/V) SDS 13 10 MG/ML SHEARED HERRING TESTIS DNA STORE AT –20°C 4 3 METHOD 1 PREPARE THE NORTHERN GEL BY DISSOLVING 3 G OF AGAROSE IN 250 ML OF DEPC TREATED WATER COOL TO 55°C AND ADD 17 5 ML FORMALDEHYDE AND 30 ML 10× MOPS BUFFER (BOTH PREHEATED TO 55°C CAST THE GEL IN AN APPROPRIATELY SIZED TRAY IN A FUME HOOD 2 BEFORE LOADING THE SAMPLES PRERUN THE GEL AT 5 V/CM FOR 5 MIN IN 1× MOPS BUFFER 3 PREPARE THE RNA SAMPLES (SEE NOTE 1) FOR ELECTROPHORESIS BY ADDING THE FOLLOW- ING TO THE RNA SAMPLE: 5 5 µL OF FORMALDEHYDE, 15 µL OF FORMAMIDE, 1 5 µL OF 10× MOPS BUFFER AND DISTILLED WATER TO A FINAL VOLUME OF 30 µL 4 DENATURE THE SAMPLE AT 55°C FOR 15 MIN AND ADD 3 µL OF LOADING BUFFER (50% GLYCEROL, 0 25% BROMOPHENOL BLUE IN DEPC-TREATED WATER) LOAD ONTO GEL AND RUN (SEE NOTE 2) 5 PLACE THE GEL IN A TRAY AND COVER WITH DEPC-TREATED DISTILLED WATER INCUBATE WITH GENTLE AGITATION FOR 15 MIN 6 REMOVE THE WATER AND REPLACE WITH 10× SSC AND SHAKE FOR 15 MIN; REPEAT ONCE 7 SET UP THE CAPILLARY BLOT AS DESCRIBED FOR SOUTHERN BLOTTING 8 AFTER TRANSFER IS COMPLETE DISMANTLE THE NORTHERN BLOT, REMOVE THE FILTER AND AIR- DRY DO NOT RINSE THE FILTER AS FOR SOUTHERN BLOTS FIX THE RNA TO THE MEMBRANE BY UV CROSSLINKING OR BAKING AT 80°C AS FOR SOUTHERN BLOTS 9 HYBRIDIZE MEMBRANE USING THE SAME CONDITIONS AS FOR SOUTHERN BLOTS 4 4 NOTES 1 THE SUCCESS OF ANY EXPERIMENTS INVOLVING RNA DEPENDS ON THE QUALITY OF THE RNA! THE MOST RELIABLE METHOD TO EXTRACT RNA FROM TISSUE SAMPLES IS TO USE A COMMERCIALLY AVAILABLE KIT 2 IF POSSIBLE, RECIRCULATE THE RUNNING BUFFER TARGET FORMAT AND HYBRIDIZATION CONDITIONS REFERENCES 1 SOUTHERN, E M (1975) DETECTION OF SPECIFIC SEQUENCES AMONG DNA FRAGMENTS SEPARATED BY GEL ELECTROPHORESIS J MOL BIOL 98, 503–517 2 SMITH, G E AND SUMMERS, M D (1980) THE BIDIRECTIONAL TRANSFER OF DNA AND RNA TO NITROCELLULOSE OR DIAZOBENZYLOXYMETHYL-PAPER ANN BIOCHEM 109, 123–132 TARGET FORMAT AND HYBRIDIZATION CONDITIONS END LABELING PROCEDURES AN OVERVIEW 1 INTRODUCTION THE PURPOSE OF THIS CHAPTER IS TO GIVE AN OVERVIEW OF THE DIFFERENT END LABEL- ING PROCEDURES THESE PROTOCOLS HAVE BEEN STANDARDIZED AND OPTIMIZED BY SEV- ERAL BIOTECHNOLOGY COMPANIES AND ARE AVAILABLE IN KITS UNLESS YOUR LABORATORY IS INVOLVED IN PRODUCING LARGE QUANTITIES OF MANY DIFFERENT PROBES (E G , MICRO- GRAMS OF EACH PROBE), IT IS UNPRACTICAL, AND PERHAPS MORE EXPENSIVE, TO SET UP YOUR OWN PROTOCOL HOWEVER, A GOOD UNDERSTANDING OF THE TYPE OF PROBE, THE LOCATION OF THE LABEL (3'-, 5'-END OR DISTRIBUTED ALL ALONG THE MOLECULE), AND THE NUMBER OF LABELED NUCLEOTIDES INCORPORATED SHOULD BE CONSIDERED WHEN PLAN- NING AN EXPERIMENT 2 LABEL LOCATION 2 1 3'-END LABELING THE EFFICIENCY OF LABELING THE 3'-END OF A DNA MOLECULE DEPENDS ON THE TYPE OF ENZYME USED, THE TYPE OF DNA MOLECULE (SINGLE- OR DOUBLE-STRANDED), AND THE LENGTH OF THE 3'-END (RECESSED, BLUNT END, OR PROTRUDING) DNA POLYMERASE I KLENOW FRAGMENT (EXO-) FILLS IN THE 3'-RECESSED ENDS OF RESTRICTION FRAGMENTS IN THE PRESENCE OF RADIOLABELED NUCLEOTIDES (1) THE NUM- BER OF LABELED MOLECULES WILL DEPEND ON THE TYPE OF LABELED NUCLEOTIDE ADDED AND THE SEQUENCE OF THE COMPLEMENTARY STRAND BLUNT END FRAGMENTS CAN ALSO BE LABELED BY REPLACING THE UNLABELED 3'-END NUCLEOTIDE BY A LABELED MOLECULE