Southern Blot Protocol

Overview

1.    Digestion of ES clone DNA with restriction enzymes and Electrophoresis of digested DNA on TBE agarose gel

2.    Transfer of DNA to nylon membrane

3.    PCR labeling of DNA probes

4.    Hybridization of radiolabeled DNA probe to membrane

1. Digestion of ES clone DNA with restriction enzymes

 

Materials

 

1.     For Digestion:

a.     Clone DNA

b.     Restriction enzymes and appropriate buffers and BSA

2.     For Electrophoresis

a.     Agarose

b.     Running buffer: 89 mM Tris borate, 2 mM EDTA, pH 8.3 (TBE)

c.      Standard DNA ladder (full scale)

d.     Digested clone DNA

 

Protocol

1.     For Digestion

a.     First, measure the concentration of DNA in clone samples.

b.     Set up a digestion reaction, using ~6 g (or as little as 2.5g) per reaction. Use a minimal volume because each reaction will need to be run on one lane of a thin gel. Typically, use 20l of DNA (6g) +5l buffer, BSA, 2l RNase A (5mg/ml), and 10-100 units of low G/C restriction enzyme. Digest at the appropriate temperature (typically 37C) for 24-48 hours. ****Before digesting your samples, optimize your digestion with non-precious DNA samples. Digested DNA should be an even, smooth streak along the entire length of the gel. You should not see a band of undigested DNA at the top. With BamHI, you may see a band at a low molecular weight, this is due to a large number of repeats containing a BamHI site in genomic DNA, and is a normal (even encouraging) occurrence.

2.     For Electrophoresis

a.     Prepare 0.7% agarose gel in TAE buffer. Make the gel as thin as possible to load your entire sample.

b.     Load small amounts of the target DNA samples (after mixing them with a suitable DNA loading buffer).

c.      As a molecular weight marker, load approx. 5-10 l of the molecular weight markers (standard or radio-labeled).

d.     Run the gel until the bands are well separated. Image the gel at this point. If you are using a standard non-radiolabeled ladder photograph a ruler with the gel, with the zero position at the wells. When you do your transfer, mark the position of the wells on the membrane with a pencil so you can determine band sizes when you complete your blot.

 

2. Transfer of DNA to nylon membrane

 

Materials

1.     Zeta-Probe membrane

2.     UV crosslinker

3.     Water bath

4.     ddH2O

5.     Depurination solution: 250 mM HCl

6.     Denaturation solution: 0.5 M NaOH, 1M NaCl

7.     Neutralization Solution: 1 M Tris-HCl (pH 7.3), 3 M NaCl

8.     20x SSC buffer: 3M NaCl, 300mM sodium citrate, pH 7.0

9.     10x SSC: 1.5 M NaCl, 150 mM sodium citrate pH 7.0

10. 2x SSC: 0.3 M NaCl, 30 mM sodium citrate, pH 7.0

11. Whatman 3MM paper

 

Protocol

1.     To depurinate the DNA prior to the transfer, do the following (this is only necessary if the hybridization target is >5 kb, which is true for some of our targets).

a.     Submerge the gel in Depurination solution (250 mM HCl), with shaking at room temperature, until the bromophenol blue marker changes from blue to yellow, about 25 minutes.

b.     Rinse the gel with sterile, double distilled water.

2.     To denature the DNA in the gel:

a.     Submerge the gel in Denaturation solution for 25 min at room temperature, with gentle shaking.

b.     Rinse the gel with sterile, double distilled water.

3.     Submerge the gel in Neutralization Solution for 25 minutes at room temperature.

4.     Equilibrate the gel for at least 10 minutes in 20x SSC

5.     Set up the blot transfer as follows, avoiding the formation of air bubbles:

a.     Place a piece of Whatman 3MM paper that has been soaked with 20× SSC atop a bridge that rests in a shallow reservoir of 20× SSC.

b.     Place the gel atop the soaked sheet of Whatman 3MM paper. Roll a sterile pipette over the sandwich to remove all air bubbles that formed between the gel and paper. Place plastic wrap to cover the entire gel and cut out the wrap around the gel such that the blot will no short-circuit.

c.      Cut a piece of Zeta-Probe Membrane to the size of the gel, with a nick in the corner for orientation.

d.     Place the membrane in ddH2O, the put it in 10x SSC for 5mins. Then place it on the DNA-containing surface of the gel. Use a pipette to eliminate air bubbles as above. If you are using a standard molecular weight ladder, be sure to mark the position of the wells on the membrane for reference.

e.     Complete the blot assembly by adding a dry sheet of Whatman 3MM paper, a 2 stack of paper towels, a glass plate, and a 200 – 500 g weight.

6.     Allow the blot to transfer overnight in Transfer Buffer (20x SSC).

7.     While the blot is still damp, fix the DNA to the blot by UV Crosslinking:

a.     Place the membrane (DNA side facing up) on Whatman 3MM paper that has been soaked in 2× SSC. Mark the side containing DNA with a pencil for reference.

b.     Expose the wet membrane to Stratalinker, 1200 for 2 minutes.

c.      Rinse the membrane briefly in sterile, double distilled water to prevent crystallization of salt from the buffer.

d.     Allow membrane to air dry.

 

3. Radioactive Probe Production

 

Materials

1.     Primers for probe PCR

2.     Template DNA (1-50 ng, optimal amount: 10 ng)

3.     Agarose mini gel

4.     Gel Extraction Kit

5.     Random 9mers (or 6mers) (50 A260/mL)

6.     NEB Buffer 2

7.     dNTPs without CTP (2.5 mM each)

8.     5uL P32 CTP (3 curries/mMol)

9.     Klenow XO- (NEB) (3-5-XO-)

 

Protocol

1.     Perform a standard PCR (50l) to make your unlabeled probe.

2.     Run PCR product on a gel, loading as much as possible.

3.     Excise band and use Gel Extraction Kit to recover your DNA. (this is necessary to ensure the specificity of your labeled probe). Quantify with a spectrophotometer

4.     Start with 30-45 ng of gel-purified probe product (1-2 L). Add this to:

      33l H2O

      2l of random 9mers (30ng).

5.     Boil this for a few minutes, then cool for less than one minute (so DNA doesnt reanneal).

6.     Quickly add:

       5 l NEB buffer 2

       5 l dNTPs (without CTP)

       5l P32 CTP

      1 l KenowXO-

7.     Incubate for 30-40 minutes at 37C.

8.     Stop reaction with 100l of 10mM EDTA, and heat at 60C for 10 minutes (to make sure that polymerase is dead).

9.     Add 17l of sonicated salmon sperm DNA (1mg/mL) and 17l spermine (100mM). Let sit for 30 min.

10. Centrifuge and remove supernatant. There should be more radioactivity in the pellet than in the supernatant (this indicates => 50% incorporation).

11. Resuspend pellet in 0.5% SDS in TE buffer.

12. Add NaOH to 0.1 M and heat to 60C for a few minutes prior to adding probe to blot in hybridization buffer. (this method produces enough probe for 2 blots).

4. Hybridization of DNA probe to membrane

 

Materials

1.     Prehybridization/hybridization solution: 

a.     50ml 7% SDS in 0.5M Na phosphate; pH 7.5

b.     50mL formamide

c.      20ml 50% dextran sulfate (Sigma 50:50 by weight)

2.     Wash buffer

a.     0.5% SDS 0.1% NaPyrophosphate in TE. Warm to 60C before use.

Protocol

1.     Place the blot into a hybridization bag (plastic bag). Heat seal around 3 sides of the blot.

2.     Add the pre-hybridization buffer. Heat-seal the bag. There should be enough buffer that the bag looks slightly puffy.

3.     Incubate the blot for at least 30 minutes at 40-50C. Place bag on the lid of a 40-50C water bath Agitate gently (by massaging) during this prehybridization step.

4.     Add NaOH to your probe to a final concentration of 0.1M and heat to 60C for a few minutes priort to adding probe to blot in hybridization buffer (using the above protocol you have produced enough probe for two blots).

5.     Change the pre-hybridization buffer for the hybridization buffer containing your probe. Incubate overnight on lid of water bath. Temperature will depend on probe (37C-65C).

6.     At the end of the incubation, cut open the bag and pour off the hybridization solution from the blot.

7.     Immediately place the membrane in a plastic tray with the 60C Wash buffer. Do three 10 min washes (all at 60C).

8.     Dry blot briefly, put into plastic wrap, and expose to BioMax film for about 22hr.