Concentration | Turkish Chemistry
May 17

PCR (polymerase chain reaction)

Let’s say you have a biological sample with trace amounts of in it. You want to work with the , perhaps characterize it by sequencing, but there isn’t much to work with. This is where PCR comes in. PCR is the amplification of a small amount of into a larger amount. It is quick, easy, and automated. Larger amounts of mean more accurate and reliable results for your later techniques.

The techniques was developed by Nobel laureate biochemist Kary Mullis in 1984 and is based on the discovery of the biological activity at high temperatures of polymerases found in thermophiles (bacteria that live in hot springs).Most polymerases (enzymes that make new ) work only at low temperatures. But at low temperatures, is tightly coiled, so the polymerases don’t stand much of a chance of getting at most parts of the .

But these thermophile polymerases work at 100C, a temperature at which is denatured (in linear form). This thermophilic polymerase is called Taq polymerase, named after Thermus aquaticus, the bacteria it is derived from.

Taq polymerase, however, has no proofreading ability. Other thermally stable polymerases, such as Vent and Pfu, have been discovered to both work for PCR and to proofread.

You’ll need four things to perform PCR on a sample:

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      1. The target sample. This is the biological sample you want to amplify from.

      2. A primer. Short strands of that adhere to the target segment. They identify the portion of to be multiplied and provide a starting place for replication.

      3. Taq polymerase. This is the enzyme that is in charge of replicating . This is the polymerase part of the name polymerase chain reaction.

      4. Nucleotides. You’ll need to add nucleotides (dNTPs) so the polymerase has building blocks to work with.

There are three major steps to PCR and they are repeated over and over again, usually 25 to 75 times. This is where the automation is most appreciated.

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          1. Annealing temperature. Starts at the low end of what you think will work, then move up as necessary. If the temperature is too low, the primers will make more mistakes and you’ll get too many bands when you run your sample on a gel. If the temperature is too high you will get no results and your gel will be blank. You want to be about 3C to 5C below the melting temperature (Tm). A rough formula for determining Tm is Tm=4(G + C) + 2(A + T).

          2. Magnesium . You want your Mg2+ to be about 1.5mM to 3mM. If you go too high, the polymerase will make more mistakes.

          3. Think carefully about primer design. Both primers should have approximately the same Tm so they both anneal at the same temperature. Two out of three bases on the 3′ end should b G or C to get good hybridization (G and C have three H-bonds so you get better polymerization). Lastly, avoid primer dimers, which occur when the primers have ends that will anneal to each other. This will produce NO product.

          4. More is not necessarily better. More polymerase produces more nonspecific product, so don’t just carelessly dump in a bunch of polymerase. Additionally, PCR reactions don’t work if there is too much .

      • 1. Your target sample is heated. This denatures the , unwinding it and breaking the bonds that hold together the two strands of the , leaving you with single stranded (ssDNA).

      2. Temperature is reduced and the primer is added. The primer now have the opportunity to bind (anneal) to the pieces of ssDNA. This labels the portions of to be amplified and provides a starting place for replication.

      3. New pieces of ssDNA are made. Taq polymerase catalyzes the generation of new pieces of ssDNA that are complimentary to the portions marked by the primers. The job of Taq polymerase is to move along the strand of and use it as a template for assembling a new stand that is complimentary to the template. This is the chain reaction in the name polymerase chain reaction.

      PCR is so efficient because it multiplies the exponentially for each of the 25 to 75 cycles. A cycle takes only a minute or so and each new segment of that is made can serve as a template for new ones.

      Perhaps the most important thing to remember is to be very aware of contamination. If, for example, you unknowingly slough off a piece of skin into your sample, then your may be amplified in the PCR reaction.  Here are some other factors to optimize your results with PCR:

      RT-PCR

      Taq polymerase does not work on RNA samples, so PCR cannot be used to directly amplify RNA . The incorporation of the enzyme reverse transcriptase (RT), however, can be combined with traditional PCR to allow for the amplification of RNA . After you add your RNA sample to the PCR machine, add a primer as usual and allow it to anneal to your target . Then add RT along with dNTPs, which will elongate the primer and make a cDNA copy of the RNA and run the PRC reaction as usual. The product of RT-PCR is a double stranded analogous to the target segment of the RNA .

May 12

Chromatography

Column chromatography is one of the most common methods of protein purification. Like many of the techniques on this site, it is as much an art form as a science. Proteins vary hugely in their properties, and the different types of column chromatography allow you to exploit those differences. Most of these methods do not require the denaturing of proteins.

To be very general, a protein is passed through a column that is designed to trap or slow up the passing of proteins based on a particular property (such as size, charge, or composition).

There are three main steps to protein purification:

    1. Capture. You need to get your protein into a concentrated form. If, for example, you are trying to isolate a protein you have synthesized in an E. coli cell, you could be looking at a protein to junk ratio of 1:1,000,000. For capture purification you need a high capacity method that is also fast. You need a speedy method because your crude solution is very likely to contain proteases in addition to your protein of interest that can quickly chew up your protein.

    2. Intermediate. Intermediate purification requires both speed and good resolution.

    3. Polishing. For the final step of purification you need a system that has both good resolution and speed. Capacity is usually irrelevant at this stage.

Some of the more common columns include:

  • IEX: Ion exchange chromatography. Good for capture, intermediate, and polish.
  • HIC: Hydrophobic interaction column. Good for intermediate purification.
  • AC: Affinity chromatography. Good for capture and intermediate purification.
  • GF: Gel filtration (size exclusion) chromatography. Good polishing step.

Let’s look at these types of columns in more detail.

Ion exchange chromatography

Ion exchange chromatography is based on the charge of the protein you are trying to isolate. If your protein has a high positive charge, you’ll want to pass it through a column with a negative charge. The negative charge on the column will bind the positively charged protein, and other proteins will pass through the column. You then use a procedure called “salting out” to release your positively charged protein from the negatively charged column. The column that does this is called a cation exchange column and often uses sulfonated residues. Likewise, you can bind a negatively charged protein to a positively charge column. The column that does this is called an anion exchange column and often uses quaternary ammonium residues.

Salting out will release, or elute, your protein from the column. This technique uses a high salt concentration solution. The salt solution will out compete the protein in binding to the column. In other words, the column has a higher attraction for the charge of salts than for the charged protein, and it will release the protein in favor of binding the salts instead. Proteins with weaker ionic interactions will elute at a lower salt, so you will often want to elute with a salt gradient. Different proteins elute at different salt concentrations, so you will want to be sure you know the properties your protein well for best results.

Also be aware that changes in pH alter the charges in proteins. Be sure you know the isoelectric point of your protein (the isoelectric point is the pH at which the charge of a protein is zero) and make sure the pH of your system is adjusted and buffered accordingly.

The basic steps in using an ion exchange column are:

    1. Prep the column. Pour your buffer over the column to make sure it has equilibrated to the required pH.

    2. Load your protein solution. Some proteins in the solution don’t bind and will elute during this loading phase.

    3. Salt out. Increase the salt concentration to elute the bound proteins. It is best to use a salt gradient to gradually elute proteins with different ionic strengths. At the end bump the system with a very high salt concentration (2-3M) to make sure all proteins are off the column.

    4. Remove salts. Use dialysis to remove the salts from your protein solution.

Temperature doesn’t have a huge effect on column chemistry. However, it is better to work cold since proteins are more stable cold.

Hydrophobic interaction chromatography

Where ion exchange chromatography relies on the charges of proteins to isolate them, hydrophobic interaction chromatography uses the hydrophobic properties of some proteins. Hydrophobic groups on the protein bind to hydrophillic groups on the column. The more hydrophobic a protein is, the stronger it will bind to the column.

Load the proteins in the presence of a high concentration of ammonium sulfate (not ammonium persulfate). Ammonium sulfate is a chaotropic agent. It increases the chaos (entropy) in , and thereby increases hydrophobic interactions (the more disordered the , the stronger the hydrophobic interactions). Ammonium sulfate also stabilizes proteins. So as a result of using an HIC column you can expect your protein to be in its most stable form.

The hydrophobic column is packed with a phenyl agarose matrix. In the presence of high salt concentrations the phenyl groups on this matrix binds hydrophobic portions of proteins. You can control elution of different column-bound proteins by reducing the salt concentration or by adding solvents.

Affinity chromatography.

Affinity chromatography relies on the biological functions of a protein to bind it to a column. The most common type involves a ligand, a specific small biomolecule. This small is immobilized and attached to a column matrix, such as cellulose or polyacrylamide. Your target protein is then passed through the column and bound to it by its ligand, while other proteins elute out. Elution of your target protein is usually done by passing through the column a solution that has in it a high concentration of free ligand.  This is a very efficient purification method since it relies on the biological specificity of your target protein, such as the affinity of an enzyme for a substrate.

Gel filtration, or size exclusion, chromatography separates proteins on the basis of their size. The column is packed with a matrix of fine porous beads.

It works somewhat like a sieve, but in reverse. The beads have in them very small holes. As the protein solution is poured on the column, small molecules enter the pores in the beads. Larger molecules are excluded from the holes, and pass quickly between the beads.

These larger molecules are eluted first. The smaller molecules have a longer path to travel, as they get stuck over and over again in the maze of pores running from bead to bead. These smaller molecules, therefore, take longer to make their way through the column and are eluted last.

May 11

Vitamin C Determination by Iodine Titration
Vitamin C (ascorbic acid) 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 acid 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 acid- titration since there are additional acids in a juice, but few of them interfere with the oxidation of ascorbic acid 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 acid:

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 concentration 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 .
  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 .
  2. Add 30 ml of 3 M sulfuric acid.
  3. Pour this solution into a 500 ml graduted cylinder and dilute it to a final volume of 500 ml with distilled .
  4. Mix the solution.
  5. Transfer the solution to a 600 ml beaker. Label the beaker as your iodine solution.

Vitamin C

 

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

Standardizing Solutions

 

  1. Add 25.00 ml of vitamin C 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 . Add distilled 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 . Strain the mixture. Wash the filter with a few milliliters of distilled . Add distilled 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 = 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

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