Acid | 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 gel 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 gel.

    3. Denature the (usually while it is still on the gel).
    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 base 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 gel 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 gel 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 11

Vitamin C Determination by Iodine Titration
Vitamin C (ascorbic ) is an antioxidant that is essential for human nutrition. Vitamin C deficiency can lead to a disease called scurvy, which is characterized by abnormalities in the bones and teeth. Many fruits and vegetables contain vitamin C, but cooking destroys the vitamin, so raw citrus fruits and their juices are the main source of ascorbic for most people.

One way to determine the amount of vitamin C in food is to use a redox titration. The redox reaction is better than an -base titration since there are additional acids in a juice, but few of them interfere with the oxidation of ascorbic by iodine.

Iodine is relatively insoluble, but this can be improved by complexing the iodine with iodide to form triiodide:

I2 + I- <–> I3-

Triiodide oxidizes vitamin C to form dehydroascorbic :

C6H8O6 + I3- + H2O –> C6H6O6 + 3I- + 2H+

As long as vitamin C is present in the solution, the triiodide is converted to the iodide ion very quickly. Howevever, when the all the vitamin C is oxidized, iodine and triiodide will be present, which react with starch to form a blue-black complex. The blue-black color is the endpoint of the titration.

This titration procedure is appropriate for testing the amount of vitamin C in vitamin C tablets, juices, and fresh, frozen, or packaged fruits and vegetables. The titration can be performed using just iodine solution and not iodate, but the iodate solution is more stable and gives a more accurate result.

Purpose

The goal of this laboratory exercise is to determine the amount of vitamin C in samples, such as fruit juice.

Procedure

The first step is to prepare the solutions. I’ve listed examples of quantities, but they aren’t important. What matters is that you know the of the solutions and the volumes that you use.

Preparing Solutions

1% Starch Indicator Solution

 

  1. Add 0.50 g soluble starch to 50 near-boiling distilled water.
  2. Mix well and allow to cool before use. (doesn’t have to be 1%; 0.5% is fine)

Iodine Solution

 

  1. Dissolve 5.00 g potassium iodide (KI) and 0.268 g potassium iodate (KIO3) in 200 ml of distilled water.
  2. Add 30 ml of 3 M sulfuric .
  3. Pour this solution into a 500 ml graduted cylinder and dilute it to a final volume of 500 ml with distilled water.
  4. Mix the solution.
  5. Transfer the solution to a 600 ml beaker. Label the beaker as your iodine solution.

Vitamin C Standard Solution

 

  1. Dissolve 0.250 g vitamin C (ascorbic ) in 100 ml distilled water.
  2. Dilute to 250 ml with distilled water in a volumetric flask. Label the flask as your vitamin C standard solution.

Standardizing Solutions

 

  1. Add 25.00 ml of vitamin C standard solution to a 125 ml Erlenmeyer flask.
  2. Add 10 drops of 1% starch solution.
  3. Rinse your buret with a small volume of the iodine solution and then fill it. Record the initial volume.
  4. Titrate the solution until the endpoint is reached. This will be when you see the first sign of blue color that persists after 20 seconds of swirling the solution.
  5. Record the final volume of iodine solution. The volume that was required is the starting volume minus the final volume.
  6. Repeat the titration at least twice more. The results should agree within 0.1 ml.
You titrate samples exactly the same as you did your standard. Record the initial and final volume of iodine solution required to produce the color change at the endpoint.

Titrating Juice Samples

 

  1. Add 25.00 ml of juice sample to a 125 ml Erlenmeyer flask.
  2. Titrate until the endpoint is reached. (Add iodine solution until you get a color that persists longer than 20 seconds.)
  3. Repeat the titration until you have at least three measurement that agree to within 0.1 ml.

Titrating Real Lemon

Real Lemon is nice to use because the maker lists vitamin C, so you can compare your value with the packaged value.

  1. Add 10.00 ml of Real Lemon into a 125 ml Erlenmeyer flask.
  2. Titrate until you have at least three measurements that agree within 0.1 ml of iodine solution.

Other Samples

 

  • Vitamin C Tablet – Dissolve the tablet in ~100 ml distilled water. Add distilled water to make 200 ml of solution in a volumetric flask. 
  • Fresh Fruit Juice – Strain the juice through a coffee filter or cheese cloth to remove pulp and seeds, since they could get stuck in the glassware. 
  • Packaged Fruit Juice – This also may require straining. 
  • Fruits & Vegetables – Blend a 100 g sample with ~50 ml of distilled water. Strain the mixture. Wash the filter with a few milliliters of distilled water. Add distilled water to make a final solution of 100 ml in a volumetric flask.

Titrate these samples in the same way as the juice sample described above.

Titration Calculations

 

  1. Calculate the ml of titrant used for each flask. Take the measurements you obtained and average them.average volume = total volume / number of trials

     

  2. Determine how much titrant was required for your standard.If you needed an average of 10.00 ml of iodine solution to react 0.250 grams of vitamin C, then you can determine how much vitamin C was in a sample. For example, if you needed 6.00 ml to react your juice (a made-up value – don’t worry if you get something totally different):

    10.00 ml iodine solution / 0.250 g Vit C = 6.00 ml iodine solution / X ml Vit C

    40.00 X = 6.00

    X = 0.15 g Vit C in that sample

     

  3. Keep in mind the volume of your sample, so you can make other calculations, such as grams per liter. For a 25 ml juice sample, for example:0.15 g / 25 ml = 0.15 g / 0.025 L = 6.00 g/L of vitamin C in that sample

May 11

Gel electrophoresis

Gel is a technique used for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric current applied to a gel matrix.[1] It is usually performed for analytical purposes, but may be used as a preparative technique prior to use of other methods such as mass , RFLP, PCR, cloning, DNA sequencing, or Southern blotting for further characterization.

The term “gel” in this instance refers to the matrix used to contain, then separate the target molecules. In most cases, the gel is a crosslinked polymer whose composition and porosity is chosen based on the specific weight and composition of the target to be analyzed. When separating proteins or small nucleic acids (DNA, RNA, or oligonucleotides) the gel is usually composed of different concentrations of acrylamide and a cross-linker, producing different sized mesh networks of polyacrylamide. When separating larger nucleic acids (greater than a few hundred bases), the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet porous matrix. Acrylamide, in contrast to polyacrylamide, is a neurotoxin and must be handled using appropriate safety precautions to avoid poisoning. Another gel matrix is agarose, long unbranched chains of uncharged carbohydrate without cross links giving a gel with large pores allowing separation of macromolecules and macromolecular complexes.

“” refers to the electromotive force (EMF) that is used to move the molecules through the gel matrix. By placing the molecules in wells in the gel and applying an electric current, the molecules will move through the matrix at different rates, determined largely by their mass when the charge to mass ratio (Z) of all species is uniform, toward the anode if negatively charged or toward the cathode if positively charged

After the is complete, the molecules in the gel can be stained to make them visible. Ethidium bromide, silver, or coomassie blue dye may be used for this process. Other methods may also be used to visualize the separation of the mixture’s components on the gel. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions. If the molecules to be separated contain radioactivity added for visibility, an autoradiogram can be recorded of the gel.

If several mixtures have initially been injected next to each other, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components.

Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are molecular weight size markers available that contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule.

Applications

Gel is used in forensics, molecular biology, genetics, microbiology and biochemistry. The results can be analyzed quantitatively by visualizing the gel with UV light and a gel imaging device. The image is recorded with a computer operated camera, and the intensity of the band or spot of interest is measured and compared against standard or markers loaded on the same gel. The measurement and analysis are mostly done with specialized software.

Depending on the type of analysis being performed, other techniques are often implemented in conjunction with the results of gel , providing a wide range of field-specific applications.

Nucleic acids

In the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to the naturally-occurring negative charge carried by their sugar-phosphate backbone.[3]

Double-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their radius of gyration, or, for non-cyclic fragments, size. Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen bonds, such as sodium hydroxide or formamide, are used to denature the nucleic acids and cause them to behave as long rods again.[4]

Gel of large DNA or RNA is usually done by agarose gel . See the “Chain termination method” page for an example of a polyacrylamide DNA sequencing gel. Characterization through ligand interaction of nucleic acids or fragments may be performed by mobility shift affinity .

Proteins

SDS-PAGE autoradiography – The indicated proteins are present in different concentrations in the two samples.

Proteins, unlike nucleic acids, can have varying charges and complex shapes, therefore they may not migrate into the polyacryl amide gel at similar rates, or at all, when placing a negative to positive EMF on the sample. Proteins therefore, are usually denatured in the presence of a detergent such as sodium dodecyl sulfate/sodium dodecyl phosphate (SDS/SDP) that coats the proteins with a negative charge.[1] Generally, the amount of SDS bound is relative to the size of the protein (usually 1.4g SDS per gram of protein), so that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or shape.[1]

Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), by native gel , by quantitative preparative native continuous polyacrylamide gel (QPNC-PAGE), or by 2-D .

Characterization through ligand interaction may be performed by electroblotting or by affinity in agarose or by capillary as for estimation of binding constants and determination of structural features like glycan content through lectin binding.

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