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now: Mon Oct 20 05:37:56 2014 ... mod: Fri Oct 5 14:15:23 2012
It's good to see smoenoe thinking it through.
- for efficient Southern detection use only positively charged nylon membrane eg. GeneScreen Plus.
- Be very careful not to physically abuse the membrane. Any scratches, scrapes, folds etc. of any kind will fail to block properly and will show a huge radioactive signal, obscuring useful information in the blot. Also be careful not to get grease from fingers, or powder from gloves on the membrane.
Preparation of Blotted Membrane
- Incubate gel to a final concentration of 1μg/ml ethidium bromide in water for at least 30 minutes with gentle agitation
- be sure to take into account the volume of the gel and the water when calculating how much ethidium to use
- for gels thicker than 6mm, increase the time for this step proportionally
- do not destain the gel
- Photograph the gel using long-wave UV (302nm - preparative setting on a transilluminator) using as brief an exposure to the additional UV light as possible to avoid over-nicking the DNA
- Expose the gel to 60 mJ/cm2 UV at 254nm (an energy setting of 600 x100μJ/cm2 on a Spectroline UV Crosslinker)
- the UV causes the ethidium to nick the DNA strands, fragmenting large pieces of DNA to sizes suitable for transfer. UV nicking is the preferred method for pulsed-field gels
- alternative nicking procedure only for standard electrophoretic gels: instead of UV exposure, nick the DNA by partial acid depurination. incubate the gel with gentle agitation in 0.25N HCl (21.5 ml concentrated HCL to 1 liter with water) for 10 minutes
- ⚠ be sure to add the acid to the water, not the other way around!
- this acid partial depurination procedure is not recommended. UV nicking is more reproducible. For pulsed-field gels, acid hydrolysis gives very inefficient transfer
- Incubate gel in denaturation solution for 30 minutes with gentle agitation.
- Rinse the gel briefly in distilled water, then incubate 30 minutes in neutralization solution.
- this brings the pH of the gel back down to neutral after the denaturation step
- Incubate the gel in 10x SSC for 15 minutes with gentle agitation
- this equilibrates the salt content of the gel closer to that of the transfer solution. If omitted, the gel can shrink and break while the transfer is happening
- Cut membrane to exact size of gel. Also cut 2 pieces of Whatman filter paper to the size of the gel, plus 2 pieces as wide as the gel, but longer (to form a wick).
- Pre-wet the membrane completely in water, then equilibrate the membrane in transfer solution.
- Fill a tray with transfer solution, place a piece of glass on the tray.
- Soak one large piece of Whatman paper in the transfer solution, place on the glass so that each end hangs down into the transfer solution, and remove bubbles by rolling with a pasteur pipette.
- Soak the second large piece of Whatman paper in transfer solution and lay on top of the first piece, again removing any bubbles by gently rolling over the paper surface with a pipette
- Place the gel on the papers on the glass, again remove bubbles.
- Cover all paper surfaces not covered by the gel with plastic wrap
- this will prevent "short-circuiting" of the transfer and ensure that all solution transfer goes through the gel (and then the membrane)
- Place the pre-wet membrane on the gel, again removing any bubbles.
- Place 2 pieces of Whatman paper pre-wet with transfer solution one-at-a-time on the membrane, removing bubbles and wrinkles for each piece.
- Place a stack of paper towels on the top Whatman papers, and apply a small weight.
- do not add a heavy weight. the goal is to ensure good contact between all the surfaces, but excessive weight can crush the gel down and inhibit transfer
- Allow to blot overnight.
- Remove the paper towels and filter paper. Mark the wells of the gel on the membrane with pencil.
- don't use pen or markers as the ink tends to dissolve in the hybridization solution
- Rinse the membrane in 2x SSC
- DO NOT ALLOW THE MEMBRANE TO DRY OUT
- The membrane can be left prehybridizing (see below) or can be left in the 2x SSC solution at room temperature if necessary. Allowing the membrane to dry out can reduce subsequent hybridization by about 5-fold, due to over-adherence of the DNA to the positively charged membrane.
- Crosslink the DNA to the membrane by exposing the damp membrane to 5 mJ/cm2 UV at 254nm (an energy setting of 50 x100μJ/cm2 on a Spectroline UV Crosslinker)
- this step is optional, but may decrease background or increase signal in some circumstances
Hybridization of DNA
- Prehybridize the membrane in hybridization solution for at least 30 minutes at 65C.
- one effective method is to prehyb with the DNA side of the membrane facing into the solution for 10 mintutes, then reverse the membrane orientation to prehybridize with the non-DNA side of the membrane facing into the solution for 10 minutes, then finally reverse orientation again and prehybridize with the DNA side of the membrane facing into the solution for at least an additional 30 minutes.
- do all of these prehybridization steps at 65C
- the goal is to block non-specific binding sites on the membrane efficiently on both sides of the membrane
- Label your probe while the membrane is prehybridizing. Use 10 - 15 ng probe per membrane if RadiolabelingDna by random priming.
- Pour off the hybridization solution and add fresh, prewarmed PEG-hyb solution.
- for large roller hybridization tubes, use 10 ml PEG-hyb. do not use an excessive volume of PEG-hyb solution
- Add the probe and hybridize at 65C overnight.
- Pour off the hybridization solution into the radioactive waste.
- Rinse membrane at 65C as follows:
- 5 minutes: rinse solution A
- 30 minutes: rinse solution A
- 30 minutes: rinse solution B
- 30 minutes: rinse solution B
- 30 minutes: rinse solution C
- Do a final brief rinse of the membrane with a 2x SSC solution (1:10 dilution of a 20x SSC stock) and wrap in Saran Wrap. Make sure the membrane is not wet (place on dry filter paper for a few minutes until all visible liquid has been absorbed) but do not allow the membrane to dry completely or it will be impossible to strip and reprobe.
- Expose and develop a series of autoradiographs.
Stripping a southern blot
If you need to strip off hybridized DNA so that a blot can be reprobed, incubate the membrane at 95C for 1 hour in 100 ml of 0.1% SDS in water (no SSC). Then begin again from the Hybridization of DNA
section above, beginning with the prehybridization step.
mix components together and heat to 50C to get the casein to dissolvestore at room temperature
- 2x SSC
- 7% SDS
- ⚠ use caution and a mask when weighing out large quantities of SDS powder. the fines are very irritating when inhaled → you should believe this
- 0.5% Hammersten casein
store at room temperature
- 2x SSC
- 7% SDS
- 0.5% Hammersten casein
- 8% PEG-6000
transfer solution - 10x SSC
Final 10x concentration is 1.5 M for NaCl and 150 mM for sodium citrate.
- Dissolve 87.7 g of NaCl and 44.1 g of sodium citrate dihydrate in 800 ml H2O.
- Adjust to pH 7.0 with HCl if necessary (but it won't in fact be necessary).
- Adjust volume to 1 liter.
- 1 liter is generally sufficient for one Southern blot
denaturation solution - 0.4N NaOH, 1.5M NaCl
per 200 ml (enough for one gel):
- 3.2 g of NaOH pellets
- 17.6 g NaCl
- bring up to 200 ml volume with water, dissolve using a stir-bar until no solid NaOH is visible
- one southern blot requires about 200 ml of denaturation solution
neutralization solution - 1M Tris pH 8.0, 1.5M NaCl
- dissolve 17.6 g NaCl in 200 ml of 1M Tris pH 8.0
- one gel requires about 200 ml of neutralization solution
- this solution must be pH 8.0. Use pH paper to check and be sure.
rinse solution A
- 2x SSC (a 1:10 dilution from a 20x SSC stock solution)
- 0.1 % SDS (a 1:100 dilution from a 10% SDS stock solution)
rinse solution B
rinse solution C
- This protocol cannot be used for NorthernBlotting since the hydroxide will destroy RNA.
- The Whatman paper can become very fragile when wet with the transfer solution. If this is a problem, do not wet the very edges of the paper and handle the wet paper by these dry edges for easier manipulations.
- It is very important for pretty results that as each layer of the southern blotting sandwich is added (wick, gel, membrane, wet filter paper) that all bubbles (including pockets of buffer) and wrinkles are carefully removed. each layer should lie completely flat upon the layer immediately below it. do not go to all the trouble to do a southern blot and then get an ugly result by scimping on this step.
- For pulsed-field gels, do not use acid depurination to nick DNA as this has been shown to reduce subsequent hybridization from pulsed-field gels when the target band is present in isolation from background non-target DNA.
- Leach TJ, Glaser RL., Quantitative hybridization to genomic DNA fractionated by pulsed-field gel electrophoresis. Nucleic Acids Res. 1998 Oct 15;26(20):4787-9.
- Lee H, Birren B, Lai E, Ultraviolet nicking of large DNA molecules from pulsed-field gels for southern transfer and hybridization. Analytical Biochemistry 1991 199:29-34.
- see http://www.millipore.com/publications.nsf/docs/tn055 for many informative technical comments
- making the hybridization solution with a final concentration of 8% PEG 6000 can greatly increase sensitivity