Lab Methods Spring 2008

From InformaticsWiki

Jump to: navigation, search

Contents

[edit] The importance of understanding laboratory methods

Laboratory methods provide the raw data used in bioinformatics analysis. The data are in public databases might result from:

  • repeated analysis and careful annotation, or
  • single-pass analysis and batch processing.

However, both kinds of data are useful for particular purposes. Therefore, the strengths and limitations of a given type of bioinformatics analysis depend on the methods used to produce the raw data. It is always important to remain aware of how the raw data are generated when performing bioinformatic analysis.

Each of the broad topics below covers multiple laboratory methods. Under each are listed are some of the most commonly used methods relevant to each topic. Each method is explained succinctly and in sufficient detail to convey its strengths and limitations, including:

  • the nature of the starting material
  • the key reagents and apparatus used
  • the nature of the basic result of the method, and
  • how that result is interpreted to produce the raw data for bioinformatic analysis.

[edit] Determining the nucleotide sequences of genes and genomes

In order to determine the nucleotide sequences of a single gene or several genes, usually we can clone the genes into certain commonly used cloning plamid, and use the dideoxy method for sequencing DNA, but now many improvement have been made to this method. DNA sequencing is completly automated. Chain-terminating nucleotides that are each labeled with a different colored fluorescent dye are used. All four synthesis reactions can be performed in the same tube, and the products can be separated in a single lane of a gel. A detector can read and record the color of the fluorescent label on each band. A computer then reads and stores this nucleotide sequences. (The general procedure of DNA clonging: amplify the target genes with PCR, digest the genes and the plasmid with appropriate restriction nuclease, join the genes into the plasmid with DNA ligase, transform the plamid into competent cells, and only the cells containing recombinant plamids can grow on antibiotic medium.) If we need to determine the nucleotide sequences of the whole genome, we need to construct a genomic DNA library. The genomic DNA is cleaved into fragments with restriction nuclease, the DNA fragments are inserted into plasmids, and the recombinant plasmids are introduced into bacteria. The entire collection of plasmids is called a genomic DNA library, and then we can use the automated DNA sequencing machine based on dideoxy method to sequence the whole genome as described above. --jinch 13:18, 17 January 2008 (EST)

Pyrosequencing is a DNA sequencing technique that is based on the detection of released pyrophosphate (PPi) during DNA synthesis. In a cascade of enzymatic reactions, visible light is generated that is proportional to the number of incorporated nucleotides. The cascade starts with a nucleic acid polymerization reaction in which inorganic PPi is released as a result of nucleotide incorporation by polymerase. The released PPi is subsequently converted to ATP by ATP sulfurylase, which provides the energy to luciferase to oxidize luciferin and generate light. Because the added nucleotide is known, the sequence of the template can be determined. Pyrosequencing has been successful for both confirmatory sequencing and de novo sequencing. This technique has not been used for genome sequencing due to the limitation in the read length, but it has been employed for applications such as genotyping, resequencing of diseased genes, and sequence determination of difficult secondary DNA structure. Reference: Mostafa Ronaghi. Pyrosequencing Sheds Light on DNA Sequencing. Genome Res. 2001 11: 3-11. --parshian

'DNA sequencing' is the determination of the precise sequence of nucleotides in a sample of DNA. The Procedure The DNA to be sequenced is prepared as a single strand. This template DNA is supplied with 

   * a mixture of all four normal (deoxy) nucleotides in ample quantities
         o dATP
         o dGTP
         o dCTP
         o dTTP
   * a mixture of all four dideoxynucleotides, each present in limiting quantities and each labeled with a "tag" that fluoresces a different color:
         o ddATP
         o ddGTP
         o ddCTP
         o ddTTP 
   * DNA polymerase I

Because all four normal nucleotides are present, chain elongation proceeds normally until, by chance, DNA polymerase inserts a dideoxy nucleotide (shown as colored letters) instead of the normal deoxynucleotide (shown as vertical lines). If the ratio of normal nucleotide to the dideoxy versions is high enough, some DNA strands will succeed in adding several hundred nucleotides before insertion of the dideoxy version halts the process.

At the end of the incubation period, the fragments are separated by length from longest to shortest. The resolution is so good that a difference of one nucleotide is enough to separate that strand from the next shorter and next longer strand. Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser beam and an automatic scanner provides a printout of the sequence.[1].--Phuong 23:24, 23 January 2008 (EST)

[edit] Methods to determine the time and place of gene expression

There are usually two methods to determine the time and place of gene expression. One method is to replace the coding portion of the gene that we are studying with a reporter gene. In most cases, the expression of the reporter gene is then monitored by tracking the fluorescence or enzymatic activity of its protein product. For example, we can replace the coding sequence of certain gene with GFP, making it under the control of the regulatory sequences of the original gene. These recombinant DNA molucules containing candidate regulatory sequences are then transfected into cells and tested for expression of GFP and get some data about the gene expression pattern of the original gene. Another method is to use in situ hybridization based on the nucleic acid hybridization which directly observes the time and place that the mRNA product of a gene is expressed. Typically, tissues or cells are gently fixed so that their RNA is retained in an exposed form that can hybridize with a labeled complementary DNA or RNA probe. In this way, the patterns of differential gene expression can be observed, and the location of specific RNAs can be determined. --jinch 13:50, 17 January 2008 (EST)

[edit] DNA Microarray

Researchers use DNA microarray technology to monitor the expression of many genes at the same time on a single chip[2]. This is useful especially in the case of disease. Gene expression from both normal and diseased cells can be analyzed on a single chip to find out if a malfunction in a particular gene is responsible for causing disease. Microarrays consist of tiny spots of DNA that are chemically linked to a glass surface. These spots are used as a template for messenger RNA and each spot represents a single gene. Messenger RNA is isolated from cells, labeled with a fluorescent tag and allowed to hybridize with the DNA on the microarray[3]. A scanner is then used to illuminate the microarray to determine the amount of gene expression. Genes that are extremely active will produce a greater fluorescence when compared to those genes with minimal activity. Genes that are inactive will show no fluorescence. When comparing diseased cells to normal cells different colored fluorescent tags are used to determine how gene expression becomes varied when cells become diseased[4]. Researchers can then use this data to isolate the genes believed to be responsible. This technology can also be used to understand how gene expression changes when dealing with different cell types. --Christina 18:50, 20 January 2008 (EST)

[edit] References

  1. ↑ http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAsequencing.html
  2. ↑ http://en.wikipedia.org/wiki/DNA_microarray
  3. ↑ http://www.genome.gov/10000533
  4. ↑ http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html

--Christina 19:04, 20 January 2008 (EST)

The microarray encompasses thousands to tens of thousands of known sequences immobilized on a microscope slide. To investigate whole-genome patterns of gene expression, the slide is hybridized with differentially labeled fluorescent RNA probes. For example the gene activity in the muscles and neurons of a nematode worm can be compared. Moreover, the total mRNA was isolated from each tissue of the worm and labeled with different dyes. The muscle mRNAs can be labeled red while the neuronal mRNAs be labeled green. These two samples are then simultaneously hybridized on the same glass containing PCR fragments representing each of the nearly 20,000 genes found in the nematode worm. When both samples hybridize to a particular spot, a yellow color is emitted. This hybridization result indicates that the particular gene is expressed in both tissues. Spots that strongly stain red correspond to genes that are mainly expressed in the muscles, but not the neurons. However, the spots that stain green represent genes that are expressed in neurons and not muscles. Moreover, microarray expression patterns are visible via grids. Each circle in the grid contains a short segment of DNA from the coding region of the specific genome the scientist is curious about.

Molecular Biology of the Gene. Fifth Edition.

[edit] Finding interactions among genes and proteins

In spite of huge diversity in terms of structure, function and localization; all cells of multicellular organisms including humans have same DNA. Hence cells obtain specific functions and properties not by changing the DNA sequences but by expressing only a particular region of DNA while repressing others. Along with general transcription factors, various non-coding(promoters, enhancers) and coding region of DNA; gene expression in eukaryotes needs several regulatory proteins that can be both DNA binding and non-binding. 8 % of the total human genes (≈ 2000) are estimated to express these DNA interacting regulatory proteins.

Identification and characterization of these DNA interacting proteins and their specific sequences in DNA was crucial in understanding various events associated with gene expression. This was facilitated by various techniques such as DNA foot printing, DNA affinity chromatography, Gel mobility shift assay,Chromatin Immunoprecipitation and so on. ----nimish 00:09, 22 January 2008 (EST)nimish 23:58, 21 January 2008 (EST)--nimish 01:10, 22 January 2008 (EST)--nimish 23:28, 22 January 2008 (EST)


There are several ways to find interactions among genes and proteins.

Two-hybrid system in Yeast can be used to reveal protein-protein interactions based on the modular nature of gene activator proteins. Using recombinant DNA techniques, two fusion proteins are created. The DNA sequence encoding for a target protein is fused with DNA that encodes the DNA-binding domain of a gene activator protein, and the DNA sequence encoding the candidate proteins is fused with the DNA that encodes the activation domain of a gene activator protein. The DNA binding domain that is fused to the target protein directs the fusion protein to the regulatory region of the a reporter genes. When this target protein binds to one candidate protein fused to the activation domain, their interaction brings together two halves of a transcriptional activator, which switches on the expression of the reporter gene. Through the data that we get about the expression of the reporter gene, we can get some idea about the protein-protein interactions.

DNA affinity chromatography is another way to determine the interaction between the DNA sequences and proteins. A DNA sequence is synthesized and linked to a porous matrix which is then used to construct a column. The total proteins of a cell is subjected to the column and the proteins that recognize the particular DNA sequence are selectively retained in the column. These proteins are then eluted from the column by salt solutions of different concentrations.

Protein affinity chromatography can identify the interaction of two proteins. A target protein is attached to polymer beads that are used as a column. When the proteins in a cell extract are washed through this column, those proteins that interact with the target protein are retained by the affinity matrix. Then we can find proteins that interact with the target proteins.

DNA footprinting is also the method that can find interactions among genes and proteins. Nucleases or chemicals randomly cleave DNA at every phosphodiester bond, but a bound regulatory protein blocks the phosphodiester bonds from attack, thereby revealing the protein's precise recognition site as a protected zone. The result can be easily shown on the SDS-PAGE gel.

There are also some other ways, such as coimmunoprecipitation, surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET),i.e. --jinch 19:59, 17 January 2008 (EST)

Gel mobility shift Assay

When a DNA binding protein binds to a DNA fragment forming DNA – Protein complex, the electrophoretic mobility of the DNA fragment in the gel is reduced. This decrement produces a shift in position of the band containing DNA binding protein. Larger the bound protein; greater is the retardation of DNA fragment. This is the basis of Gel mobility shift assay.

In this assay, a short DNA fragment of known sequence and size is first labeled radioactively. The DNA fragments are then mixed with cell extract. The mixture is then loaded on to a polyacrylamide gel and subjected to electric field. If the cell extract comprises proteins binding to that particular sequence of DNA; series of bands are observed on the autoradiography of the gel. The bands are retarded based on the size of protein attached to DNA. On the same gel, DNA fragments that are not mixed with lysate are also run simultaneously. The autoradiography of the DNA sample with and without bounded protein is thus compared. Using gel mobility shift assay, various sequence specific interactions between DNA and DNA binding protein can be explored. --nimish 01:05, 22 January 2008 (EST)


[edit] References

Molecular Biology of the cell.Fifth edition

Kuby immunology.Fourth edition

[edit] Various laboratory methods utilizing gel electrophoresis

Electrophoresis is the migration of charged molecules in solution in response to an electric field. Their rate of migration depends on the strength of the field; on the nett charge, size and shape of the molecules and also on the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used analytically to study the properties of a single charged species, and as a separation technique.

Support Matrices

Generally the sample is run in a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run: at the end of the run, the matrix can be stained and used for scanning, autoradiography or storage.

In addition, the most commonly used support matrices - agarose and polyacrylamide - provide a means of separating molecules by size, in that they are porous gels. A porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely. Because dilute agarose gels are generally more rigid and easy to handle than polyacrylamide of the same concentration, agarose is used to separate larger macromolecules such as nucleic acids, large proteins and protein complexes. Polyacrylamide, which is easy to handle and to make at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation.

Separation of Proteins and Nucleic Acids

Proteins are amphoteric compounds; their nett charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a nett negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The nett charge carried by a protein is in addition independent of its size - ie: the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules.

Nucleic acids however, remain negative at any pH used for electrophoresis and in addition carry a fixed negative charge per unit length of molecule, provided by the PO4 group of each nucleotide of the the nucleic acid. Electrophoretic separation of nucleic acids therefore is strictly according to size. SDS- PAGE OF PROTEINS

Separation of Proteins under Denaturing conditions

Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by "wrapping around" the polypeptide backbone - and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length - ie: the denatured polypeptides become "rods" of negative charge cloud with equal charge or charge densities per unit length. It is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size: this is done with 2- mercaptoethanol or dithiothreitol. In denaturing SDS-PAGE separations therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight.[5]--Phuong 01:20, 25 January 2008 (EST)

Southern Blot

Researchers utilize Southern blotting when they need to detect the presence of a gene that was transformed into a mixed cell population. Procedure: 1) DNA (genomic or other source) is digested with a restriction enzyme (commonly EcoR1) and separated by gel electrophoresis, usually an agarose gel. Because there are so many different restriction fragments on the gel, it usually appears as a smear rather than discrete bands. The DNA is denature into single strands by incubation with NaOH. 2) The DNA is transfered to a membrane which is a sheet of special blotting paper. The DNA fragements retain the same pattern of separation they had on the gel. 3) The blot is incubated with many copies of a probe which is single-stranded DNA. This probe will form base pairs with its complementary DNA sequence and bind to form a double-stranded DNA molecule. The probe is either radioactive or has an enzyme bound to allow visibility. 4) The location of the probe is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen or gives off light which will expose X-ray film. If the probe was labeled with radioactivity, it can expose X-ray film directly [6].

Northern Blot

A northern blot is very similar to a Southern blot (hence the name) except it is RNA instead of DNA that is "digested" (procedure "1)" from southern blot). Messinger RNA (mRNA) is the specific type of RNA isolated in Northern blotting procedures [7].

Northern Blots can also be used to determine the size of the mRNA template, identify variations which occur within the gene, and measure the relation of species. --Pittser 21:56, 23 January 2008 (EST)


Western Blot

Western blotting is a technique used to identify and locate proteins based on their ability to bind to specific antibodies. This method is very useful when there is a large mixture of proteins. Using northern blotting, the size and expression of the protein of interest is revealed [8]. Procedure: 1) Proteins are separated by gel electrophoresis (usually SDS-PAGE). 2) The proteins are transfered to a sheet of special blotting paper called nitrocellulose, though other types of paper, or membranes, can be used. The proteins retain the same pattern of separation they had on the gel. 3) The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. An antibody is then added to the solution which is able to bind to its specific protein. The antibody has an enzyme or dye attached to it which cannot be seen at this time. 4) The location of the antibody is revealed by incubating it with a colorless substrate that the attached enzyme converts to a colored product that can be seen and photographed [9]. --Ron 18:04, 21 January 2008 (EST)


Polymerase Chain Reaction (PCR)

PCR is the method to amplify specific DNA sequences. The primary materials or reagents that are used in PCR are: DNA nucleotides, the building blocks for the new DNA, template DNA, the DNA sequence that you want to amplify, primers that are required for initiation of DNA synthesis, oligonucleotides, DNA polymerase (Taq Polymerase is an enzyme derived from the bacterium Thermus aquaticus), a heat stable enzyme that catalyzes the synthesis of new DNA. Also, buffer solution is required for providing a suitable chemical environment for optimum activity and stability of the DNA polymerase and divalent cations are used. There are three steps in PCR: 1)Denaturation at around 94 degree celsius. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA. 2) Anealing at around 54 degree celcius, allowing annealing of the primers to the single-stranded DNA template. 3) Extension at around 72 degree celcius. DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTP's that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. During final elongation step, single step is occasionally performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.[10]. Every cycle results in a doubling of the number of strands DNA present. With PCR it is possible to amplify a single piece of DNA, or a very small number of pieces of DNA, over many cycles, generating millions of copies of the original DNA molecule. PCR is a powerful technique in medical diagonostics, forensics, and molecular evolution. PCR can provide valuable diagnostic information in medicine. Bacteria and Viruses can be readily detected with the use of specific primers. Also, DNA analysis can be used to establish guilt in criminal cases.[11]. To verify whether the PCR generated the anticipated DNA fragment, agarose Gel Electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a molecular weight marker, which contains DNA fragments of known size, run on the gel alongside the PCR products.[12]--Patels19 21:37, 25 January 2008 (EST)


Restriction fragment length polymorphism (RFLP)

RFLP is the identification of specific restriction enzyme that reveal a pattern difference between the DNA fragment sizes in individual organisms. DNA from an individual specimen is first extracted and purified. Purified DNA may be amplified by polymerase chain reaction (PCR). Then restriction enzymes (RE) are used to cut DNA at specific recognition sites to discover RFLP. Sample DNA is cut with one or more RE’s and resulting fragments are separated according to molecular size using Gel Electrophoresis. Electrophoresis separates the DNA molecules based on their molecular weight. Differences in fragment length can result from base substitutions, additions, deletions or sequence rearrangements within RE recognition sequences.[13]. For example, screening human DNA for the presence of potentially deleterious genes in sickle cell gene. Sickle cell disease is a genetic disorder in which both genes in the patient encode the amino acid valine (Val) in the sixth position of the beta chain (betaS) of the hemoglobin molecule. However, normal beta chains (betaA) have glutamic acid at this RFLP can provide valuable information in screening sickle cell gene.[14].--Patels19 19:29, 23 January 2008 (EST)

[edit] Macromolecular structure determination

The macromolucular structure can be determined through X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. In x-ray crystallography, x-ray is directed at a sample of a pure macromolucular crystal, most of the x-ray pass straight through it. A small fraction are scattered by the atoms in the sample. The scattered waves reinforce one another at certain poinits and appears as diffraction spots when recorded by a detector. Deducing the three-dimensional structure of a large molecule from the diffraction pattern of its crystal is a complex task and is done by computer. Unlike x-ray crystallography, NMR does not depend on having a crystalline sample. It simply requires a small volume of concentrated macromolecular solution that is placed in a strong magnetic field. NMR can provide data about the distance between the parts of the macromolecule. By combining this information with other available information, it is possible to compute the three-dimensional structure of the macromolecule. --jinch 20:59, 17 January 2008 (EST)

Mass spectrometry is another commonly used method for determining the characteristics of a protein.

[edit] Mass spectometry

The technique was first used by J.J Thomson in his vacuum tube during the beginning of the century. In his book entitled "Rays of Positive Electricity and Their Application to Chemical Analysis" it was noted that mass spectometry could be used to determine the number of various isotopes, the quantity of each kind of isotope and lastly the size. Mass spectometry is used for the recognition of specific compounds while also determining their chemical properties and structure. A minimal concentration of 10^-12 g to that of 1000 Daltons can be recognized. While this technique can not give the molecular mass directly it can give the mass to charge ratio. Originally scientists were only able to use gases to determine the ratio however with new advancements liquids and solids can be used as well. [15]

This figure which was obtained from the American Society for Mass Spectometry illustrates the features of the mass spectrometer http://www.asms.org/whatisms/images/fig1.gif --Pittser 21:17, 23 January 2008 (EST)

MS gives possibility to analyze protein complexes providing you with protein-candidates for the complex of interest. One of the widely used method of mass spectrometry (MS) is a Matrix - Assisted Laser Desorption Ionization Time of Flight analizer or MALDI - TOF. This method is more gentle compared to the regular MS. The major difference is in the special crystalline matrix on which protease cleaved proteins are applied. This matrix protects polypeptides from destructive influence of laser beam (337 nm). Also matrix facilitates evaporation and ionization of applied polypeptides. Polypeptides upon laser excitation acquire different charges depending on its amino acid sequence and length. These charged polipeptides than separated by TOF mass analyzer depending on its mass-to-charge ratio (m/z). Light ions will reach detector first. Acquired data from detector is than analyzed by specially designed program that compare results with existing database. As a result you receive the list of proteins with the sequence similarity in percents of possible proteins that was analyzed. This is considerably rough data that given to scientist to perform further investigation on the exact content of the analyzed proteins using other methods of protein - protein interaction analyses previously mentioned on this page. --User:Khakhisv 9:17, 24 January 2008 (EST)

[edit] How to build a gel electrophoresis machine

This all comes from a magazine I have. It is listed here for reference [16] Make magazine

First we will extract some DNA from our cheek cells. Mix 3 buffer solutions as follows:

1. Rinsing buffer- 1.5g (1/4tsp) table salt. 5g (1tsp) of baking soda. 1/2 cup bottled water.

2. Running buffer- .05g table salt (a pinch). 2g (1/2tsp) of baking soda. Up to a liter of bottled water. We want the buffer to measure a ph of 7.5, add water to lower the ph, or ad baking soda to raise it.

3. Loading buffer- Use 1.25ml (1/4 tsp) glycerol/glycerine and several drops of red food coloring.

First extract the DNA by swirling 5ml (1tsp) of rinsing buffer around in your mouth for 30 sec. Spit the buffer into a paper cup and pour it into a test tube. Squirt a bit of liquid soap (1/4tsp) into your sample and mix. Slowly add 5ml (1tsp) of cold rubbing alcohol to the sample. Pour it at an angle so you form 2 undisturbed layers of liquid. After 10 minutes, the DNA should appear as a whitish, snot like substance floating between the rinse solution layer and the alcohol.

Now we make the gel box. The Gel electrophoresis process lets you separate and visualize different DNA molecules based on their sizes. Agar Gel is a dense microscopic network of sugar. It is difficult for the lager DNA molecules to travel through. But because DNA is negatively charged you can draw it through the Gel by applying an electric field. A Gel box uses this principle to act as a DNA racetrack. The shorter the DNA strand is the faster it runs through.

The box consist of 2 nested containers. An inner casting chamber contains the Gel itself and this sits inside an other buffer chamber which contains the running buffer. Two opposite ends of the casting chamber are open to give the Gel direct contact with the buffer solution. We use two electrodes connected to 9 volt batteries to electrify the buffer chamber. We will use half of a plastic travel soap dish for the inner casting chamber. This will sit inside of a larger rectangular Tupperware container for the other buffer chamber. We then use the lid of a plastic slide box inside a box made of Lego blocks. This is lined with wrap to prevent leakage. I am not sure what this is used for.

We then prepare the Gel. The Gel needs two wells at one end in which to insert the DNA. We can make these by casting the Gel around a 2-pronged Lego comb sticking down into the casting chamber. Seal the open ends of the casting chamber with masking tape. Heat 8g (2tsp) of agar and 125ml (1/2cup) of running buffer in a small pot. Stir until the agar is fully dissolved and the solution is clear. Then pour this liquid into your Gel casting chamber to a thickness of between 0.5cm to 1cm. Hang the comb into the Gel at one end and let the Gel cool. After it sets remove the comb and the masking tape.

We then prepare the DNA by using a wooden toothpick to spool up the DNA from the extraction step. We then mix 7 parts of rubbing alcohol to 3 parts of water in a small container. We then dip the DNA snot on the toothpick into this solution for a few seconds. We then air dry this for 10 minutes. Then we scrape this off into 75ul (3 droplets ) of running buffer. We allow this to dissolve overnight at room temperature. We then add 1 drop of loading buffer. Your sample is now ready to run.

We now run the Gel. We will load the DNA samples into the wells of the Gel. We then place the Gel casting chamber into the buffer chamber and cover it with running buffer until it is fully immersed with 0.5cm of fluid above the Gel. We then connect the electrodes to 5-7 9 Volt batteries connected in series. Since the DNA is negatively charged make sure that the positive electrode is inserted at the end opposite of the wells. The batteries should apply enough power for a 3 hour run. The exact time will depend on the dimensions of the Gel and the buffer chamber. The smaller the chambers the faster the run due to less resistance. You should see bubbles forming at the positive electrode.

After the run we will stain the Gel. We will need to make a 0.02% solution of Methylene Blue in distilled water. Then immerse your Gel chamber into this solution overnight at room temperature. If it works right you should be able to see at least one faint band of DNA from your cheek cells.

This DNA finger print process is not good enough to match the sample with the person who submitted it but it can probably be used to determine if the DNA came from a human verses a banana. You should be able to take DNA from your cheek and a banana and compare them using this method.

I have pictures of this machine and how to build it if anyone is interested. The web site has a picture of the device also. [17]

John Keubler --johnnyk 21:37, 23 January 2008 (EST)

[edit] Collaborating With The World On Your Findings

All of the methods above strengthen our resolve is displaying that lab work is the building block of science. However, it is equally important to realize that all of us scientists are able to do what we do because we are standing on the shoulders of giants. The fastest way to make this happen in 2008 is to launch our research into the world wide web.

By integrating the research of the individual cell with the entire body of knowledge we are able to expedite research in every facet of science. One of such great tools is the NCBI Tax[18]. When one learns to appreciate the multi-functionality and raw power that this scientific tool has to offer, more doors open to them then they could imagine.

In fact, the hole NCBI site is full of information on genes, proteins, nucleotides as well as dozens of tools to learn more about each[19].--Alex Shalman 22:01, 23 April 2008 (EDT)

References:

  1. ↑ http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/D/DNAsequencing.html
  2. ↑ http://en.wikipedia.org/wiki/DNA_microarray
  3. ↑ http://www.genome.gov/10000533
  4. ↑ http://www.ncbi.nlm.nih.gov/About/primer/microarrays.html
  5. ↑ http://www.mcb.uct.ac.za/sdspage.html
  6. ↑ http://www.bio.davidson.edu/COURSES/GENOMICS/method/Southernblot.html
  7. ↑ http://www.escience.ws/b572/L13/north.html
  8. ↑ http://www.molecularstation.com/protein/western-blot/
  9. ↑ http://www.bio.davidson.edu/COURSES/genomics/method/Westernblot.html
  10. ↑ http://en.wikipedia.org/wiki/
  11. ↑ Berg, Tymoczko, and Stryer. BIOCHEMISTRY: 5th Edition. New York: W. H. Freeman and Company, 2001.
  12. ↑ http://en.wikipedia.org/wiki/
  13. ↑ http://www.iscid.org/encyclopedia/Restriction_Fragment_Length_Polymorphism
  14. ↑ http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RFLPs.html
  15. ↑ http://www.asms.org/whatisms/p4.html
  16. ↑ Backyard Biology Make: Vol 7
  17. ↑ http://makezine.com/07/fingerprinting
  18. ↑ Browserhttp://www.ncbi.nlm.nih.gov/sites/entrez?db=taxonomy
  19. ↑ http://www.ncbi.nlm.nih.gov/About/index.html
Retrieved from "http://tyrosine.umdnj.edu/wiki/index.php/Lab_Methods_Spring_2008"
Personal tools