Analysis of nucleic acids

Analysis of Nucleic Acids 1

Southern Blot

Digest approximately 15µg of genomic DNA sample with appropriate restriction enzyme and

appropriate molecular weight. Run a gel at 25V for 15 minutes and then at 100V (It is also

possible to run the gel o/n at 20-25V), stain with Ethidium bromide and take a picture with a

ruler (put the 0 at the slots)

Transfer protocol 1

1) - Rinse the gel <in distilled water and place it in a clean dish containing 0.25M HCl.

Shake slowly on a platform shaker for 30min.

HCl provoke partial depurination of DNA which in turn leads to strand cleavage. This

allows a better transfer of large DNA restriction fragments.

2) - Pour off HCL and rinse with distilled water. Add denaturing solution and shake slowly

for 30min.

Denaturation unzips the DNA to give single-stranded molecules that have unpaired bases

suitable for further hybridization.

3) - Pour off denaturing solution and rinse with distilled water. Add neutralization solution

and shake slowly for 30min.

The aim of neutralization solution is to bring pH above 9. At higher pH, DNA will not bind

to the membrane.

4) - Over a plastic or glass dish, place a glass support. Cut a piece of Whatman paper which

width is similar to that of the gel and which length is longer than the support glass

support. Fill in the dish with transfer buffer (20X, 10X, 5X SSPE or 20X SSC), wet the

Whatman piece and place it so that each extremity are submerged in the transfer buffer.

This will constitute the bridge to generate capillarity.

5) - Place the gel over the bridge and squeeze out air bubbles with a glass pipette. Cut 4

pieces of plastic wrap and place over the gel edges.

This is to prevent the buffer from “short circuiting”, i.e. so that it flows through rather

than around the gel.

6) Cut a piece of nylon membrane just large enough to cover the exposed surface of the gel.

Pour transfer buffer in a glass dish and wet the membrane by placing it at the surface of

Analysis of nucleic acids

Analysis of Nucleic Acids 2

the liquid. Place the wetted membrane on the surface of the gel. Try to avoid getting air

bubbles under the membrane; remove any that appear by carefully rolling a glass pipette

over the surface.

7) Place 4-5 Whatman pieces on the membrane and stack paper towel on the top. Lay a glass

plate on the top of the structure and put a weight on the top. Leave overnight.

Transfer protocol 2 (vacublot)

8) - Cut a membrane so that it is a bit bigger than the plastic seal. Place the plastic seal and

lock the system. Take the gel and place it onto the membrane in the vacublot system.

Remove bubbles by squeezing them with a glass pipette. Switch on the vacuum pump.

9) - Incubate with each of the solutions discarding the previous one before adding the next.

10) - Cover the gel with 20X SSPE and wait for 90min checking every 30min the buffer level

11) - Dismount the system and take the membrane.

Vacuum pump

gel Membrane Plastic wrap

Analysis of nucleic acids

Analysis of Nucleic Acids 3

Common things in both protocols:

- A correct transfer is achieved when all the loading blues have transferred on the membrane.

- Once the transfer done, cross-link the membrane under UV light. Dry and keep dry until use.

- Wash everything after use, salts crystallize.

Breaking solution (0.25M HCl) (1L):

Fuming HCl 19.2ml

Denaturing solution (1.5M NaCl, O.5M NaOH) (1L): 5M NaCl 300ml

NaOH 20g

Neutralization solution (1.5M NaCl, 0.5M Tris· HCl pH 7.0) (1L): 5M NaCl 300ml

1M Tris· HCl pH 7.0 500ml