Biochemistry | Turkish Chemistry
May 14

Southern blotting

Southern blotting

Southern blotting was named after Edward M. Southern who developed this procedure at Edinburgh University in the 1970s. To oversimplify, are transferred from an agarose onto a membrane. Southern blotting is designed to locate a particular sequence of within a complex mixture. For example, Southern Blotting could be used to locate a particular gene within an entire genome.

The amount of needed for this technique is dependent on the size and specific activity of the probe. Short probes tend to be more specific. Under optimal conditions, you can expect to detect 0.1 pg of the for which you are probing.

This diagram shows the basic steps involved in a Southern blot.

dSouthern blot

Let’s look at this technique in greater detail.  

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    • 1. Digest the with an appropriate restriction enzyme.

    2. Run the digest on an agarose .

    3. Denature the (usually while it is still on the ).
    For example, soak it in about 0.5M NaOH, which would separate  double-stranded into single-stranded . Only ssDNA can transfer.

    A depurination step is optional. Fragments greater than 15 kb are hard to transfer to the blotting membrane. Depurination with HCl (about 0.2M HCl for 15 minutes) takes the purines out, cutting the into smaller fragments. Be aware, however, that the procedure may also be hampered by fragments that are too small.

    Be sure to neutralize the acid after this step, or the after the prior step if you don’t depurinate.

    Transfer DNA to membrane4. Transfer the denatured to the membrane. Traditionally, a nitrocellulose membrane is used, although nylon or a positively charged nylon membrane may be used. Nitrocellulose typically has a binding capacity of about 100µg/cm, while nylon has a binding capacity of about 500 µg/cm. Many scientists feel nylon is better since it binds more and is less fragile. Transfer is usually done by capillary action, which takes several hours. Capillary action transfer draws the buffer up by capillary action through the an into the membrane, which will bind ssDNA.

    You may use a vacuum blot apparatus instead of capillary action. In this procedure, a vacuum sucks SSC through the membrane. This works similarly to capillary action, excepts more SSC goes through the and membrane, so it is faster (about an hour). (SSC provides the high salt level that you need to transfer .)

    After you transfer your to the membrane, treat it with UV light. This cross links (via covalent bonds) the to the membrane. (You can also bake nitrocellulose at about 80C for a couple of hours, but be aware that it is very combustible.)

    5. Probe the membrane with labeled ssDNA. This is also known as hybridization.
    Whatever you call it, this process relies on the ssDNA hybridizing (annealing) to the on the membrane due to the binding of complementary strands.
    Probing is often done with 32P labeled ATP, biotin/streptavidin or a bioluminescent probe.

    A prehybridization step is required before hybridization to block non-specific sites, since you don’t want your single-stranded probe binding just anywhere on the membrane.

    To hybridize, use the same buffer as for prehybridization, but add your specific probe.

    6. Visualize your radioactively labeled target sequence. If you used a radiolabeled 32P probe, then you would visualize by autoradiograph. Biotin/streptavidin detection is done by colorimetric methods, and bioluminescent visualization uses luminesence.

32P labeled ATP
Treat the dsDNA fragment that you are using as a probe with a limiting amount of Dnase, which causes double-stranded nicks in . Add 32P, dATP, and other dNTPs to polymerase I, which has 5′ to 3′ polymerase activity and 5′ to 3′ exonuclease activity.

Nick translation occurs and as the nick is translated down the strand, the polymerase activity continues to nick while the exonuclease activity continues to fill in the nick. As this happens, 32P becomes incorporated into, and thus labels, the . Heat the to make it single stranded, then immediately place it on ice to keep the two strands from reannealing to each other. (If the is on ice, the passes through the annealing temperature too quickly for the to rehybridize into double-stranded .)

May 14

Agarose gel electroporesis

Agarose gel electroporesis

Agarose gel electrophoresis separates DNA fragments according to their size. Typically, a DNA molecule is digested with , and the agarose gel electrophoresis is used as a diagnostic tool to visualize the fragments. An electric current is used to move the DNA molecules across an agarose gel, which is a polysaccharide that functions as a sort of sieve to help “catch” the molecules as they are transported by the electric current.

This technique has lots of applications. Generally speaking you can analyze DNA fragments that result from an enzyme digestion of a larger piece of DNA to visualize the fragments and determine the sizes of the fragments. In addition to its usefulness in research techniques, agarose gel electrophoresis is a common forensic technique and is used in DNA fingerprinting.

The phosphate molecules that make up the molecules have a high negative charge. When DNA is placed on a field with an electric current, these negatively charged DNA molecules migrate toward the positive end of the field, whichin this case is an agarose gel immersed in a buffer bath. The agarose gel is a cross-linked that is somewhat like a three-dimensional or screen. The DNA molecules are pulled to the positive end by the current, but they encounter from this agarose . The smaller molecules are able to navigate the faster than the larger one, so they make it further down the gel than the larger molecules. This is how agarose electrophoresis separates different DNA molecules according to their size. The gel is stained with ethidium bromide so you can visualize how these DNA molecules resolved into bands along the gel.

Southern blotting may also be used as a visualization technique for agarose gels.


Unknown DNA samples are typically run on the same gel with a “ladder.” A ladder is a sample of DNA where the sizes of the bands are known. So after you run out your sample, you can compare the unknown fragments to the ladder fragments and determine the approximate size of the unknown DNA bands by how they match up to the known bands of the ladder.

May 12

Determination of Vitamin C in A Table

Vitamin C (L-ascorbic acid) has received much attention lately, as a result of claims that it can cure various diseases, ranging from the common cold to . It is known that vitamin C is an antioxidant and is required for . It is also used for treatment of rheumatoid arthritis. Vitamin C is readily oxidized by iodine in an acidic solution

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