Turkish Chemistry

Alkaline lysis is the method of choice for isolating circular plasmid DNA, or even RNA, from bacterial cells. It is probably one of the most generally useful techniques as is a fast, reliable and relatively clean way to obtain DNA from cells. If necessary, DNA from an alkaline lysis prep can be further purified.

Alkaline lysis depends on a unique property of plasmid DNA. It is able to rapidly anneal following denaturation. This is what allows the plasmid DNA to be separated from the bacterial chromosome.

Typically, you will grow up E coli cells that contain the plasmid you want to isolate, then you will lyse the cells with alkali and extract the plasmid DNA. The cell debris is precipitated using SDS and potassium acetate. This is spun down, and the pellet is removed. Isopropanol is then used to precipitate the DNA from the supernatant, the supernatant is removed, and the DNA is resuspended in buffer (often TE). A mini prep usually yields 5-10 ug. This can be scaled up to a midi prep or a maxi prep, which will yield much larger amounts of DNA (or RNA).

Specific protocols for alkaline lysis differ widely from lab to lab, and even from scientist to scientist. The basic principles behind the procedure, however, are fairly uniform. Here they are:

1. Spin down your cells

2. Discard the supernatant. Pieces of cell wall are released from the bacteria and are floating around in the supernatant. These cell wall pieces can inhibit enzyme action on your final DNA, so it is important to get rid of all of the supernatant and to even invert the tube and wipe the lip with a Kim-wipe or Q-tip.

3. Resuspend the cells in buffer (often Tris) and EDTA. EDTA chelates divalent metals (primarily magnesium and calcium). Removal of these cations destabilizes the cell membrane. It also inhibits DNases. Glucose should also be added to maintain osmolarity and prevent the buffer from bursting the cells.

4. Lyse the cells with sodium hydroxide (NaOH) and SDS. This highly alkaline solution gave rise to the name of this technique. Mix this by gentle inversion and incubate on ice for five minutes (but no longer, or your DNA will be irreversibly denatured). Three things happen during this stage:

a. SDS pops holes in the cell membranes. SDS (sodium dodecyl (lauryl) sulfate) is a detergent found in many common items such as soap, shampoo and toothpaste.

b. NaOH loosens the cell walls and releases the plasmid DNA and sheared cellular DNA.

c. NaOH denatures the DNA. Cellular DNA becomes linearized and the strands are separated. Plasmid DNA is circular and remains topologically constrained.

5. Renature the plasmid DNA and get rid of the garbage. Add potassium acetate (KAc), which does three things:

a. Circular DNA is allowed to renature. Sheared cellular DNA remains denatured as single stranded DNA (ssDNA).

b. The ssDNA is precipitated, since large ssDNA molecules are insoluble in high salt.

c. Adding sodium acetate to the SDS forms KDS, which is insoluble. This will allow for the easy removal of the SDS from your plasmid DNA.

Now that you’ve made it easy to separate many of the contaminants, centrifuge to remove cell debris, KDS and cellular ssDNA. Your plasmid DNA is in the supernatant, while all of the garbage is in the pellet.

6. Precipitate the plasmid DNA by alcohol precipitation (ethanol or isopropanol) and a salt (such as ammonium acetate, lithium chloride, sodium chloride or sodium acetate) and spin this down. DNA is negatively charged, so adding a salt masks the charges and allows DNA to precipitate. This will place your DNA in the pellet.

7. Rinse the pellet—your plasmid DNA—in ice-cold 70% EtOH and air-dry for about 10 minutes to allow the EtOH to evaporate.

8. Resuspend your now clean DNA pellet in buffer (often Tris) and EDTA plus RNases to cleave any remaining RNA. Your DNA is now back in solution.

DNA of this purity is good for a number of uses, such as in vitro transcription or translation or cutting with some enzymes. If you are sequencing or transforming this DNA into mammalian cells, you’ll want to use additional purification techniques such as phenol extraction, Qiagen column purification, or silica-based purification. 

A Hexagonal Molecule on which is Founded Aromatic Chemistry

Benzene is one of the most interesting organic chemicals of all because it took so long for chemists to understand the structure. It was a mystery for so long but once its structure was determined it proved to be the key to a host of large molecules.

Discovery
The famous chemist, Michael Faraday discovered benzene in 1825 while distilling crude oil. Its empirical formula was found but its structure was not determined until 1865 when German chemist, Friedrich August Kekulé had a day-dream or “reverie” while thinking about the structure. He saw a snake curl round and bite its own tail, forming a ring structure. Kekulé immediately knew that benzene was a ring of six carbons connected by alternating double and single bonds (see image 2)

Hybridisation
The problem with this structure was that double and single bonds have different lengths, but X-ray crystallography studies have shown that the bond-lengths in benzene are the same. This led some scientists to suggest that the structure resonated between double and single bonds. Eventually Linus Pauling used his molecular orbital theories to propose a structure in which all the bonds are equal with a hybridised circular orbital above and below the benzene ring (see image 1).

Arenes
Benzene is the simplest of the group of organic chemicals known as arenes. The hydrogen atoms attached to each carbon in the ring can be substituted for other functional groups to form other chemicals many of which are important in industry for example. Benzene rings can be fused together to make structures such as naphthalene. Graphite is made up of a whole hexagonal network of carbon atoms in which all the hydrogen atoms have been replaced by other carbon atoms.

Lead-Free Petrol
One of the most common uses of benzene is as an additive to petrol or gasoline in cars. It improves what is known as the octane rating and reduces knocking. In the drive to reduce lead-containing additives in petrol benzene has been used as a replacement, although there are some health issues related to benzene itself. It is used in many parts of the chemical industry as a raw material in the manufacture of plastics, lubricants, drugs and pesticides.

Carcinogen
In the early twentieth century benzene was used as an after-shave lotion because of its pleasant smell, but it has since been discovered to be seriously hazardous to the health. It can be carried through the blood and damage bone marrow and red blood cells. It is also carcinogenic and can cause leukaemia.
The copyright of the article The Chemistry of Benzene in Organic Chemistry is owned by Simon Davies. Permission to republish The Chemistry of Benzene in print or online must be granted by the author in writing.

Molecular Size

Alternative fuels tend to be made up of small, fairly simple molecules; for example, here are schematic chemical diagrams (C denotes a carbon atom, H is hydrogen, and O is oxygen) of
methane (CH₄), the primary constituent of liquefied or compressed natural gas, and
propane (C₃H₈), the primary constituent of liquified petroleum gas.

Petroleum fuels are blends of lots of different chemical species; in general, the molecules of a liquid petroleum fuel are pretty big and complex. Here is
isooctane (C₈H₁₈), typical of the molecules found in gasoline (I had to spread out the structure a bit to get all the hydrogen atoms to fit in the picture–all of these molecules are, of course, three-dimensional, but some squish into a plane better than others!), and

this monster is cetane, or n-hexadecane (C₁₆H₃₄), typical of diesel fuel.

Incomplete Combustion

When a hydrocarbon fuel (that is, one that is made up of hydrogen and carbon) burns completely, the oxygen in the air combines with the hydrogen to form water (H₂O) and with the carbon to form carbon dioxide (CO₂). If the burning is not complete, then some of the carbon atoms only combine with one oxygen atom rather than two, to form carbon monoxide (CO), a highly poisonous gas.

Some of the carbon atoms may remain stuck together with each other and with some of the hydrogen atoms as well, so that unburned hydrocarbon molecules (mostly smaller than the ones in the original fuel) can also come out the tailpipe. These unburned hydrocarbons (plus any fuel hydrocarbons that evaporate from the fuel system before getting into the engine to be burned at all) react with nitrogen oxides (another pollutant from combustion) in the presence of sunlight to form ozone, which is a lung irritant (the “ozone layer” in the stratosphere is a shield against the sun’s ultraviolet light, but at ground level ozone is the main component of “photochemical smog”). Carbon atoms can also remain stuck to one another with few or no hydrogen atoms attached, especially during incomplete combustion of diesel fuel, producing soot.

This is one of the reasons alternative fuels are less polluting than gasoline and diesel: their simpler molecules are easier to burn more completely in an engine, so that less carbon monoxide, soot, and unburned hydrocarbons come out the tailpipe. In addition, any unburned hydrocarbons that are produced are less reactive than those that come from incomplete burning of gasoline or diesel fuel, and so they produce less ground-level ozone; methane in particular is almost incapable of forming smog.

Oxygen Content

Some alternative fuels are not hydrocarbons; alcohols and biodiesel contain oxygen atoms as well as carbon and hydrogen. Here are the chemical structures of the common alcohol fuels,
methanol (CH₃OH) and
ethanol (C₂H₅OH).
(Biodiesel molecules are “monoalkyl esters”, but I haven’t been able to trace down anything more specific. The “ester” part of that name, however, indicates that the molecules include oxygen atoms.)

In many parts of the USA, gasoline is “oxygenated” during at least part of the year; this means that oxygen-bearing compounds are added to the fuel mixture. The reason for doing this is that having some oxygen as part of the fuel molecules to start with promotes more complete combustion, so that less carbon monoxide, soot, and unburned hydrocarbons come out the tailpipe, as described above. Alcohol fuels and biodiesel carry this one step further, in that the oxygen-bearing compound is not an additive at the 5 to 10 percent level, but a major constituent of the fuel, which increases the benefits of oxygenation.

Carbon Content

Even if, with the aid of electronic engine controls and efficient catalytic converters, a hydrocarbon fuel is burned completely to water and carbon dioxide, there is now growing concern about carbon dioxide as a greenhouse gas. Measures to cut back on production of carbon dioxide by automobiles without sacrificing performance can focus on efficiency, i.e., getting as much useful propulsive power out of a given amount of fuel as possible, which typically involves replacing the traditional drivetrain of a piston engine driving the wheels through a gearbox with a more efficient design.

However, some fuels inherently produce less carbon dioxide when burned completely than gasoline or diesel fuel. For example, counting the numbers of oxygen atoms it takes to burn up an isooctane molecule and a methane molecule (typical of gasoline and natural gas respectively), one can calculate that 100 oxygen atoms will combine with four isooctane molecules to produce 32 carbon dioxide molecules and 36 water molecules, while the same number of oxygen atoms will combine with 25 methane molecules to produce 25 carbon dioxide molecules and 50 water molecules. That is, a given amount of air (oxygen) will produce about 25% less carbon dioxide if used to burn natural gas than if used to burn gasoline. (Of course, this advantage will be reduced if you have to open the throttle wider and burn an additional amount of air with natural gas to get the same amount of power, but in the real world the 25% figure turns out to be about right.)

Avoiding Carbon Dioxide Emissions Entirely

The other thing to consider is the source of the carbon in the fuel; if it came from the carbon dioxide in today’s air to begin with, like an alcohol fuel produced by fermenting biomass (as opposed to a fossil fuel, whose carbon came out of the air when the dinosaurs were around!), then returning it to the air now adds nothing to the net flow of carbon dioxide into the atmosphere. Alcohol fuels or biodiesel produced from plants, when burned, just return to the air the carbon dioxide that those plants took out of the air while growing.

Finally, there’s one fuel that, in itself, produces no carbon dioxide at all when burned, namely
hydrogen; there’s no carbon there to produce carbon dioxide!
Of course, since free hydrogen molecules don’t occur in nature, it is typically produced by “reforming” a hydrocarbon or alcohol fuel or by using electricity to split water into hydrogen and oxygen. Then the size of the contribution of hydrogen fuel to carbon dioxide emissions depends on the source of the hydrocarbon fuel that was reformed or the source of the electricity used to split the water.

If a fossil fuel was the ultimate source of the energy that is, in effect, stored in the hydrogen, then you can still gain a large improvement in carbon-dioxide production if the hydrogen is used in an efficient drivetrain, as noted above; the same is true for the electrical energy stored in a battery-powered electric vehicle. In order to obtain the full benefits of reduction of carbon dioxide (or of ordinary air pollutants like carbon monoxide), of course, the energy used to split the hydrogen or charge the battery can be obtained from a renewable source like wind power or photovoltaics.

The nice thing about hydrogen- or battery-powered vehicles is that they can run on whatever is available–efficient natural-gas-burning powerplants today, with an increasing contribution from renewable energy as time goes on and the price of photovoltaic cells (solar cells) and other renewable energy sources continues to decline. As renewable energy becomes an ever larger part of the power generation mix over the next few decades, hydrogen- and battery-powered vehicles can switch over to the new power sources without a hiccup–it’s all electricity to them!