SOUTHERN BLOTS

CIPITATION: Quick spin DNAThen, add 245l Mix of salt & H20 (200l H2O + 45l 3M Na Acetate): Add 2l glycogenFill eppendorf to top with 100% EtOHPpt either at -20° C overnight or in dry ice for 30min or -70° C for 30min (STABLE---can store)3. WASH:Vortex 5secSpin for 30 min at 1350 rpm in cold roomDiscard EtOH.Add 70% EtOH to 1/2 fill eppendorf. Vortex 5sec.Spin 10 min at 1350 rpm. Discard EtOHQuick spin. Remove excess remaining EtOH using P200 pipette.Air Dry 10 minAdd 20l 1X loading buffer (Stock 6X)Heat 15-30 min at 50° CLoad 20l /well. First vortex, then quick spin each sample.4. PREPARING GELS: While samples are spinning, prepare gels (Vertical Hoefer apparatus, 3mm thick gels):APPARATUS ASSEMBLY: Take 2 clean glass plates. Put white marker and spacer on each end (3mm), then place on flat area and tighten. Place plates in apparatus and tighten with black plugs. Place 1/2 in tape around top of 3mm comb to decrease well size and place comb in apparatus.PLUG PREPARATION: Prepare 5cc per gelBis-acrylamide (19.5:1) : 2.5ccH2O : 2.5ccTEMED : 15l APS :150l Pour 4cc in each apparatus. It will solidify in 3-5min.GEL PREPARATION: Prepare 1% agarose gel in 1X TAE. Use 1g agarose in 100cc 1X TAE (without EB). Microwave. Cool to 80-90° C. Pour 100cc rapidly into apparatus. Use a transfer pipette (with top & bottom cut-off) to facilitate rapid pouring.. Add additional solution for next 5minutes around comb sites to prevent bubbles. Cool at room temperature for 1hour.Remove comb gently out of gel apparatus by gently sliding back and forth. Clear top of each apparatus and remove excess gel by using a scalpel and clean each well with a toothpick.Assemble gel for Southern by placing buffer well on top of gel and tightening with black plugs. Pour 1X TAE buffer in well box and make sure it does not leak.Remove gel from stand and place in Hoefer apparatus. Fill Hoefer with 1X TAE buffer.5. GEL LOADING:Load 20l / well. First vortex then quick spin each sample before loading6. ELECTROPHORESIS: Run at 45V for 20minutes, then 80V for 1hour and then 90V until samples have run about 9cm from well. Total running time: app 3hours7. STAINING: Remove gel from apparatus. Mark lower right-sided corner for gel#1 and top & lower right-sided corner for gel #2.Stain with ethidium bromide solution for 10 minutes in 500cc solution (100l in 1L) Discard and rinse with H2O.Expose film in dark room with eagle eye. Get picture with ruler on left side next to 1kb marker.Trim marker and excess edges out under UV light.8. HCL WASH:Wash in HCL (0.1M) for 10min exactly. DiscardRinse with H2O. Put in 0.4M NaOH solution9. TRANSFER:Prepare transfer apparatus. Remove paraffin. Pour freshly-prepared NaOH solution half way up the apparatus. Remove all airbubbles from apparatusCut 3mm Whatman paper-5-6pcs per gel as well as Zetaprobe nylon membrane to size of the gel. Rinse nylon membrane in waterTransfer in the following sequence: 3mm filter paper(soaked in NaOH)-------Gel-----------------Nylon membrane (presoaked in H2O)-----------2-3pcs 3mm filter paper (soaked in NaOH)------------2 pcs dry filter paperAt each step remove all air bubblesPut stack of paper towels and heavy book on top of transfer membraneTransfer overnightIn the morning, remove dry filters. Remove nylon with gel and trim excess nylon off edges. Cut right-hand corners of nylon membrane according to the gelWick in 2X SSC buffer for 5minutesRinse with H2OPut nylon membrane as sandwich between two pieces of dry 1.5 mm filter paperBake in oven for 30minutes at 80° C to crosslink DNA. Label each membraneIF STORING, THEN WICK IN 2X SSC FOR 3MIN AND THEN SARAN WRAP AND STORE AT RT10. PREHYBRIDIZATION:Survey Hot Room with Geiger CounterStart oven at hybridization temperature, usually 60° C Thaw blotto by gentle heating in crock pot in hot room or 80° ovenPrepare nylon membrane: i) either crosslink or ii) if used before must strip membrane with Southern wash 2 before reusingWick membrane in 2X SSC for 5minutesRoll each nylon membrane away from you and place in hybridization bottle. Use 2X SSC to unroll the membranes Discard 2X SSC and put 20cc Blotto. Prehybridize for >1hr, usually 2hr (upto 48hr)11. PROBE PREPARATION:Use 0.5ml tubePROBE: Probe (> 100ng/l ): x l H20: (23-x) l Heat 95° for 5min then on ice for 1 mindATG mix: 6 l (dATP,dGTP,dTTP mix)10X Hex buffer: 4 l Klenow: 1.5 l 32P-dCTP: 5 l Incubate probe at 37° C for 30-45 minutes12. PROBE PURIFICATION:Use Pharmacia S200 Microspin columnsPlace each column in a 2.0 ml eppendorf tube. Spin for 2.5min at 3000rpmSpin the probe for 2.5 min at 3000rpmAdd 60l column elution buffer to the probe. Add hot probe to the column and spin for 2.5 min at 3000rpmRemove column carefully. Check activity of eluted probe---should be hot!Add another 100l elution buffer to the eluateAdd 2l of probe to scintillation vial (1:100 dilution) and measure 32P activity (Program 3). Final activity is : measured CPM X 100Boil probe for 3minutes and then on ice for 1minRemove prehybridization buffer from hybridization bottle and add probe with Blotto (Total vol blotto: 15ml)Hybridize overnight in oven at the desired temperature, usually 60° C13. SOUTHERN WASH:Prewarm wash buffers at 65° C in hot room (Southern wash solutions 1 & 2)Decant the hybridization buffer in liquid radio wasteRinse with 20ml Wash 1 solution. Decant into liquid wasteWash with 2/3volume Wash 1 solution for 10minutes at 65° C X 2. Discard in sink Rinse with 20ml Wash 2 solution. Discard in sinkWash with 2/3 volume Wash 2 so[1] [2] 下一页
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