Quantitative Estimation of DNA Concentrations

DNA, RNA, and protein strongly absorb ultraviolet light in the 260 to 280 nm range. UV spectroscopy can be used as a quantitative technique to measure nucleic acid concentration and protein contamination. Nucleic acids strongly absorb at 260 nm and less strongly at 280 nm while proteins do the opposite. The general rules for determining the concentrations of nucleic acids at 260 nm are:

1. 1 Optical Density (OD) unit of double-stranded DNA is 50 micrograms/ml.
2. 1 OD unit of single-stranded DNA is 33 micrograms/ml.
3. 1 OD unit of single-stranded RNA is 40 micrograms/ml.

Proteins absorb strongly at 280 nm where 1 OD unit is 1 mg/ml. When using UV spectroscopy for estimating DNA concentrations, it is very important to remove all protein and RNA from the DNA solution. Good estimations can only be made on clean preparations.

An estimate of the purity of a DNA preparation can be made by measuring the absorbance at both 260 nm and 280 nm. Pure solutions of nucleic acid will absorb approximately twice as much at 260 nm as at 280 nm. Experimentally, the ratio of 260 nm/280 nm of a pure DNA solution is between 1.8 to 2.0. As protein contamination increases, the ratio decreases. Additionally, the presence of contaminating oranic solvents, such as phenol, can affect estimations of concentration and purity.

Materials you need are:

UV Spectrophotometer

Quartz or UV compatible cuvettes

TE buffer

DNA template

Method:

1. Fill the cuvette with water or TE buffer. Zero the spectrophotometer at 260 nm with this blank.
2. DNA from plasmid and genomic preparations is typically at a concentration exceeding 1 micrograms/microliter. Consequently, DNA is usually diluted before measuring its absorbance. An unfortunate result of this measurement is that the DNA is expended as a result of the dilution. Be sure these is adequate DNA to waste. Start by diluting the DNA sample 1 microliter : 999 microliters of TE buffer (the dilution can be done directly in the cuvette). Mix the dilution thoroughly.
3. Measure the optical density (OD). Multiply the resulting OD by 50 micrograms/ml. For a 1:1000 dilution, the mass of DNA is equal to micrograms/microliter.
4. Similarly, the same sample can be measured at 280 nm. A ratio of the OD-260nm/OD-280nm is an indicator of DNA purity. A ratio of 1.8 or higher indicates minimal protein contamination.

Isolation Of Genomic DNA Rrom Yeast

Isolating genomic DNA from yeast involves culturing the microbe, harvesting the cell, enzymatically removing the cell wall, lysing the protoplast, and finally separating the DNA from the other cell debris.
Materials that you need are:

Yeast culture-prepared previously

Spectrophotometer with cuvettes

50 mM EDTA, pH 8-ice cold

50 mM Tris, pH 9.5, 2% 2-mercaptoethanol

1.2 M sorbitol, 50 mM Tris, pH 7.5

Lyticase solution-500 U/ml in 50 mM Tris, pH 7.5

10% Sodium Dodecyl Sulfate (SDS)-used for checking protoplast formation

Lysis buffer-100 mM Tris, pH 7.5, 100 mM EDTA, 150 mMNaCl, 50 micrograms/ml RNase A

Lysis buffer with 2% SDS

95% Ethanol-stored at minus 20 degree Celcius

TE buffer-10 M Tris, pH 8, 1 mM EDTA

3 M potassium acetate, pH 5.5

Here are the step by step methods:

1. The yeast can be cultured for as long as 48 hours at 30 degree Celcius. The optical density of a 1:10 dilution of the culture in water can be as high as 1.0 at 520 nm.
2. Harvest 5 ml of cells by centrifugation (5 minutes at 5000 rpm). Resuspend the yeast in 1 ml of cold 50 mM EDTA, pH 8, and transfer to a 1.5 ml microfuge tube. Centrifuge for 1 minute, decant, and resuspend again in 50 mM EDTA.
3. Pellet the cells as before and suspend the cells in 1 ml of 50 mM Tris, pH 9.5, 2% 2-mercaptoethanol. Incubate for 10 min at room temperature. Centrifuge and decant.
4. Resuspend the cells in 800 micro liter of 1.2 M sorbitol, 50 mM Tris, pH 7.5. The sorbitol act as an osmotic support and prevents rupture of the cells as the wall is removed. As the yeast cell walls degrade, membranes can easily overextend and rupture.
5. Add 200 micro liter of Lyticase (500 U/ml in 50 mM Tris, pH 7.5). Place the cells on a rocker and incubate at 37 degree Celcius for one hour. Lyticase is a yeast cell wall degrading enzyme isolated from the bacteria Arthrobacter luteus.
6. Examine the suspension under a microscope to ensure protoplast formation. As the yeast wall is degraded, the cell membrane can ooze out of the sack. Viewed with phase contrast microscopy, yeast protoplasts are characteristically refractile (or bright) spheres, and yeast cell wall shells appear as gray ghosts (cell walls without membrane and cytosol). Combine 10 micro liter of 10% SDS with 10 micro liter of yeast protoplasts. Examine the cells under the microscope. The absence of refractile yeast indicates the protoplasts were lysed by the SDS.
7. Pellet the protoplasts by centrifuging at 10000 rpm for five minutes. Resuspend the cells in 1 ml of 100 mM Tris, pH 7.5, 100 mM EDTA, 150 mM NaCl (lysis buffer). Transfer the cells to a 5 ml polypropylene tube. Add 1 ml of lysis buffer with 2% SDS. Mix and incubate at 30 Celcius egree for 30 minutes. Check the cells under a microscope for lysis.
8. Centrifuge the lysate at 5000 rpm for 15 min to pellet cellular debris. Decant the upper phase containing the DNA.
9. Using a pipet, determine the volume of the DNA solution. Add 1/10th volume (e.g., 100 micro liter for every ml) of 3 M potassium acetate to the solution. In the presence of ions Na and K, DNA precipitates if mixed with either ethanol or isopropanol. Incubate the DNA at -20 Celcius degree for 30 min (or overnight if possible. Centrifuge the solution at 7000 rpm for 20 minutes. The DNA appears as white pellet. Decant and remove as much moisture as possible, but do not allow the pellet to dry. Once genomic DNA drys, it can be very difficult to resuspend.
10. Resuspend the DNA in 100 micro liter of TE buffer and freeze.

Gold Nanoparticles In Cancer Cell Detection

“Gold nanoparticles are very good at scattering and absorbing light,” said Mostafa El-Sayed, director of the Laser Dyanamics Laboratory and chemistry professor at Georgia Tech. “We wanted to see if we could harness that scattering property in a living cell to make cancer detection easier. So far, the results are extremely promising.”

Many cancer cells have a protein, known as Epidermal Growth Factor Receptor (EFGR), all over their surface, while healthy cells typically do not express the protein as strongly. By conjugating, or binding, the gold nanoparticles to an antibody for EFGR, suitably named anti-EFGR, researchers were able to get the nanoparticles to attach themselves to the cancer cells.

“If you add this conjugated nanoparticle solution to healthy cells and cancerous cells and you look at the image, you can tell with a simple microscope that the whole cancer cell is shining,” said El-Sayed. “The healthy cell doesn’t bind to the nanoparticles specifically, so you don’t see where the cells are. With this technique, if you see a well defined cell glowing, that’s cancer.”

In the study, researchers found that the gold nanoparticles have 600 percent greater affinity for cancer cells than for noncancerous cells. The particles that worked the best were 35 nanometers in size. Researchers tested their technique using cell cultures of two different types of oral cancer and one nonmalignant cell line. The shape of the strong absorption spectrum of the gold nanoparticles are also found to distinguish between cancer cells and noncancerous cells.

What makes this technique so promising, said El-Sayed, is that it doesn’t require expensive high-powered microscopes or lasers to view the results, as other techniques require. All it takes is a simple, inexpensive microscope and white light.

Another benefit is that the results are instantaneous. “If you take cells from a cancer stricken tissue and spray them with these gold nanoparticles that have this antibody you can see the results immediately. The scattering is so strong that you can detect a single particle,” said El-Sayed.

Finally, the technique isn’t toxic to human cells. A similar technique using artificial atoms known as Quantum Dots uses semiconductor crystals to mark cancer cells, but the semiconductor material is potentially toxic to the cells and humans.