Neutral Southern with Alkaline Transfer

Obtained from Lisa Patek, Miller Lab 10/7/11

Materials

      Amersham Rediprime Random Prime Labeling System

      Whatman 3mm Chr Filter Paper (Cat. 3030 917)

      Office Depot Single Fold Paper Towels

      32P-labeled dCTP (Perkin Elmer BLU513H250UC)

      Kodak BioMax XAR Film (Cat 165 1579)

      Illustra Autoseq G-50 columns (GE Healthcare 28917925, also available at UW Biochem Store)

      Salmon Sperm DNA (Sigma D1626) see attached preparation protocol

      Positively charged nylon membrane (BioRad Zeta Probe GT Genomic Tested)

      Extra thick blotting paper (i.e. for semi-dry transfer)

Solutions

Depurination Solution (made fresh)

      0.25N HCl (21.6mL concentrated HCl in final volume 1L ddH2O

Transfer Buffer (made fresh)

      16g NaOH/1L ddH2O

 

0.2M Tris pH 7, 2x SSC

      For 250ml: 50ml 1M Tris pH 7, 25ml 20x SSC, and 175ml H2O.

 

Phosphate Buffer

      1 M NaHPO4 pH 7.2

o   134 g Na2HPO4-7H2

o   4 ml 85% H3PO4 (concentrated phosphoric acid) 

o   Bring to 1L with dH2O, adjust pH to 7.2

Church Buffer

      1% BSA, 1 mM EDTA, 0.5 M NaPO4 pH 7.2, 7% SDS

o   500ml phosphate buffer pH 7.2

o   10 g BSA fraction V (crystalline grade Sigma A-4503) 

o   2 ml 0.5 M EDTA 

o   70 g SDS

Membrane Wash #1

      100ml 2xSSC, 0.1% SDS

o   10ml 20x SSC

o   1ml 10% SDS

o   89ml ddH2O

Membrane Wash #2

      150ml 0.2x SSC, 15ml 10% SDS, 133.5ml ddH2O

o   1.5 mL 20x SSC

o   15mL 10% SDS

o   133.5ml ddH20

 

Protocol

Preparation of DNA

1.    Digest 1-10g of genomic DNA overnight.

2.    Run on 0.7-0.8% 1xTAE gel for 4-6 hours or overnight.

 

Acid Depurination (only necessary for fragments larger than 4kb)

1.    Remove gel to UV light box and photograph with ruler.

2.    Transfer gel to clean Tupperware and add 500ml depurination solution. Let rock until dyes change color (to yellow). Replace with 500ml fresh depurination solution and rock for an additional 15 minutes.

 

Alkaline Transfer (start here if not doing acid depurination)

1.    Prepare blotting paper, filter paper, and paper towels by cutting to the exact size of the gel. Cut the membrane barely larger than the gel. For the wick, create an overhang on all four sides (about an inch) so that the buffer will flow from all four sides, not just the top and bottom. Cut an additional sheet of blotting paper that is the same size as the gel casting tray.

2.    Invert a clean, dry casting tray in a large Tupperware container or developer tray. Set the wick on the casting tray and fold down the edges over the sides of the tray. Wet the wick with the 0.4M NaOH and roll out the bubbles using a 10ml pipet. Pour some NaOH on the wick and add the sheet of blotting paper that is the size of the tray; roll out bubbles. Pour NaOH on top, carefully transfer gel on top of blotting paper; roll out bubbles. Pour NaOH on the gel and add the membrane to the stack. Pour NaOH onto the membrane and add the piece of blotting paper cut to the size of the gel (be careful that the blotting paper does not overhang the membrane and touch the gel), roll out bubbles. Note that you should not roll directly on the membrane. Add the thicker blot paper and the paper towels on top (about 3-4 inches). Do not weigh the stack down with glass or a weight. Check the stack for overhangs that will short-circuit the transfer.

3.    Add remaining NaOH to Tupperware—usually almost up to the level of the gel. Allow to transfer overnight.

4.    Once the transfer is complete remove all paper towels and blotting paper. In the upper right corner of the membrane (opposite of the DNA) label the blot (using a pencil). Draw a line marking the position of the wells for reference.

5.    Remove the membrane to a piece of Whatmann blotting paper (DNA side up). Place the blotting paper to a Tupperware container with 250ml of 0.2M Tris pH 7, 2x SSC (50ml 1M Tris pH 7, 25ml 20x SSC, and 175ml H2O).

6.    If you want to crosslink the membrane use the autocrosslink function on the Stratalinker.

 

Prehybridization

 

1.    Pre-warm a clean hybridization cylinder in over set at 65C, and warm the Church buffer to 65C.

2.    Boil salmon sperm DNA for 5-10 minutes then cool quickly on ice.

3.    Add 15ml Church buffer to hybridization cylinder plus Salmon Sperm DNA (the amount in 1 eppendorf) at a final concentration of 150g/ml. Rotate the cylinder containing the buffer and SS DNA in the 65C oven until the membrane is ready.

4.    Add the membrane to the cylinder by rolling it up lengthwise, try to remove all bubbles between the membrane and the cylinder (if you are starting with a dry membrane, if you crosslinked it you should wet it in Tris wash buffer or 2x SSC).

5.    Prehybridize for 2 hours to overnight.

 

Hybridization

 

1.    Add 25ng of gel purified probe (from serial PCRs) to an Eppi tube, bring volume up to 45l with TE.

2.    Boil 5 minutes and snap cool on ice.

3.    Transfer probe to tube of Rediprime, do not mix at this point.

4.    Add 5l (50Curies) of P-32 alpha dCTP to the probe/Rediprime tube and mix by pipetting up and down. Incubate at 37C for 15 minutes.

5.    While the probe incubates prepare a G-50 spin column (all spins 2 minutes at 800xg) by removing preservative and washing with 250l of H2O.

6.    After probe is done at 37C transfer to the top of dry spin column and spin.

7.    Boil probe 5 minutes and snap cool on ice.

8.    Add to prehybridization solution in the cylinder with blot. Hybridize overnight, 65C.

 

Membrane Wash

 

1.    Pour hybridization solution into hot waste.

2.    Rinse blot with 10ml Solution 1, pour into hot waste.

3.    Add 45ml Solution 1 and incubate at 65C in oven for 15 minutes. Pour off in sink.

4.    Add remaining 45mls Solution 1. Incubate 15 minutes at 65C, then pour off into sink.

5.    Rinse blot with 10ml, prewarmed Solution 2, pour off into sink.

6.    Add 45ml Solution 2 and incubate at 65C for 30 minutes. Pour off into sink.

7.    Remove blot with forceps to Tupperware with remaining 65C solution 2. Wiggle rinse.

8.    Transfer blot to 3MM paper until puddles gone (not bone dry) and wrap in plastic wrap.

9.    Expose to X-ray film overnight or for several days.

 


 

 

Salmon Sperm DNA Preparation

Obtained from Lisa Patek, Miller Lab 10/7/1010

 

1.    Dissolve salmon sperm DNA (Na salt Sigma D1626) in ddH2O at 10mg/ml. Stir at room temperature to help dissolve if desired.

2.    Extract once with phenol and once with phenol:chloroform.

3.    Pull the aqueous phase and shear by rapidly passing the DNA through an 18g needle twelve times.

4.    Precipitate the DNA by adding 2 volumes of ice cold ethanol (you may need to add sodium acetate).

5.    Spin to pellet and dissolve at a concentration of ~10mg/mL in ddH2O.

6.    Check the OD and calculate the concentration.

7.    Boil for 10 minutes and store at -20C in small aliquots.

 

Before use boil for 5-10 minutes and snap cool on ice. Use in prehybridization solution at 150g/mL