12/5/2023 0 Comments Southern blot hybridizationRemember, the notched corner is where your ladder is! Recipes For 1 Liter of 20xSSPE Store at -80☌ ON (at least 15 hours) - may take longer for good exposure. Wrap in saran wrap and pop it in film cassette with intensifying screen. Mount membrane on filter paper (tape it down). If you have tried this protocol with your probe before and gotten only a weak signal, try omitting Wash 4 and/or 5. Gently polish membrane with kimwipe after last wash to lower background discard kimwipe in radioactive waste. (in Tuperware, discard down drain, noting on sink's radiation log) (in Hyb Oven tube, discard wash in Radioactive waste) Washes 1-3 are in 1X SPPE, 1% SDS, 30' each. Remove Hyb buffer (discard in radioactive waste). Make Hybridization Buffer (FBI+hot Probe).Īdd 1 × 106 cpm/mL of denatured probe (denature at 100 ☌ for 5', secure the top of tube with parafilm or cap lock) to 5-10 mL of pre-heated FBI buffer (5 × 106 to 1 × 107 cpm total).ĭiscard "old" prehyb buffer, add hot hyb buffer and incubate at 65☌ for at least 6 hours (ON good). It is always a good idea to do this fresh! 6. Once heated and in solution, add 20 mL FBI buffer to 30 cm tube Hyb oven tube. If you have FBI on your self, heat now to 55-65 ☌. Make up FBI buffer! (also called pre-hybridization buffer at this stage) Keep it warm. Also, no need to crosslink! Transfer in basic solution takes care of that. Membrane can also be stored in saran wrap in a refrigerator for a while at this stage. This isn't important, but it helps if the membrane is a little dry to get into hybridization oven tube. Neutralize blot in 100 mL neutralization buffer - 15' or so.ĭry membrane - putting it between two pieces of Whatman paper is good. Let go for at least 4-6 hours, though overnight is great. A smooth piece of plastic with a heavy book (or 2) on top works well.Īdd plenty of 0.4 N NaOH to the tray. Sheets of Parafilm to surround gel like a frame and prevent wicking.Do not move the membrane around once settled on gel. For further orientation, you can mark the membrane with pencil or VWR marker. Line up top of the membrane to the wells of the gel, with cut corners in the same quadrant. Hybond-N+ membrane (GE Biosciences) (pre-soaked in 0.4 N NaOH, top right corner removed for orientation).(Now the cut corner is on the top right.) Two sheets of Whatman filter paper cut slightly larger than gel size.Sponges/towels must be larger than the gel. Big sponges or paper towels (2-3 inches in height) in 0.4 N NaOH.The sandwich - in a big tray set up the following from the bottom up: (Add base to the water, not the other way around. Be careful! Lower % gels love to crack.Īdd 500 mL of 0.4 N NaOH to neutralize (480 mL H2O plus 20 mL 10 N NaOH). After the gelĪdd 500 mL of 1:50 dilution of HCL (490 mL H2O, then add 10 mL HCl) to depurinate. Once gel is finished running, be sure to take a picture with a ruler that glows under UV!!! (Line-up zero on ruler with wells).Ĭut off top, left corner of gel (near your marker) for future orientation. You can also run overnight at 20 to 25 volts. Add ethidium to gel and ALLl running buffer. The gel should be very long (25-40cm) to give good separation of bands. TAE is better than TBE for resolving larger fragments. Pour low percentage (usually 0.6% to 0.8%) agarose gel in 1X TAE. This is a good time to make 20xSSPE, 1M Tris pH 7.5 and TAE if you don't have them already. ![]() Too much will overwhelm the signal of the real bands.ĭigest 6 hours to overnight (usually ON is the easiest/best). If using plasmid DNA or BAC DNA, use 1 ng or LESS. It is always a good idea to run a control. Spermadine can be purchased from GE Biosciences, #US21760 Southern Blots with Alkaline Transfer Procedure 1.
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