Labs 6, 7 and 8:
Plasmid DNA extraction from E. coli, Restriction Enzyme Digestion Laboratory,
and Polymerase Chain Reaction Laboratory
The purpose of lab six was to extract plasmid DNA from E. Coli, which is then used as the template DNA material being cut by restriction enzymes later in lab seven. Additionally, it serves as a template for the amplification of the ampicillin resistance gene in lab eight. Agarose gel (1%) electrophoresis was enacted in both labs to determine the sizes of cut and amplified DNA fragments.
There are two methods to excise bacterial plasmid DNA. A Rapid Extraction Procedure for Screening Recombinant DNA reitorates these methods. The first involves the lysing of bacterial cells, followed by centrigugation to remove the majority of the chromsonal DNA, and then banding of remaing DNA in a cesium chloride gradient in the prescence of ethidium bomide. This yields a highly purified concentration, however, for electrophoresis, an extremely pure solution of DNA is not needed, therefore the second method is preferred. The second and more rapid method for preparing DNA plasmid is alkaline extraction, where a colony of cells are lysed and plasmid DNA an be detected in crude extractment processes to be scanned by electrophoresis in 1% agarose gel.
The purpose of agarose gel electrophoresis is to separate DNA fragments to help determine sizes of plasmid DNA, sizes of fragments cut by restriction enzymes, and sizes of amplified genes that come from a polymerase chain reaction.
Restrcition enzyme digestion enables DNA to be recombined by cutting plasmid DNA and inserting additional DNA fragments cut from the same enzymes. The newly formed DNA will then express both genes from the plasmid DNA and fragmental DNA. PCR is the process of replicating templates of DNA,
It was hypothesized that as sizes of DNA fragments decrease, due to digestion by restction enzymes or PCR, the distance traveled by the banded fragments within the gel will increasing significantly. This hypothesis is supported by observations that the smaller a DNA fragment is (less mass) the band will travel at a faster rate and reach a closer relative distance to the positive pole of the electric field.
METHODS AND MATERIALS:
The materials used in lab six through eight are as follows: E. coli, restriction enzymes, solutions to extract and prepare plasmid DNA, 1 % agarose gel electrophoresis, molecules involved in a (PCR) polymerase chain reaction, and a thermal cycler.
To prepare a pure enough DNA strand so it can be used in PCR or restriction enzyme digestion, the cell must go through several processes to have its DNA excised correctly. E. Coli was the bacteria used to obtain a certain piece of plasmid DNA called PGEM-HIS1. Eight steps encompassed this experiment for extracting plasmid DNA from E. Coli. The first step involved the denaturiton of cell walls by resuspending the E. Coli into 100 microliters of solution constituted of 25 microliters of Tris, 50 microliters of Glucose, and 10 microliters of EDTA. EDTA’s purpose was to protect the DNA from digestion from DNAases by binding to Mg2+ (Cofactor of DNAases) Inhibiting it’s function , and Tris’ purpose was to act as a buffer and keep the PH around 8.0. This solution in lab six was called Solution 1. The second step was the addition of Solution 2, which was a 200 microliter solution made up of 1% SDS and .2 N NaOH (strong base). SDS’s purpose was to break up the cell proteins and plasma membrane. NaOH’s purpose was to initially rupture the cell can cause the solution to become extremely basic. This basic solution forced the DNA to dilute into two linear strands. The third step, addition of 150 micrliters of Solution 3, involved 5M glacial acetic acid and 3M potassium acetate. Acetic acid in solution three neutrailized the PH of the solution and allow the renaturiation of DNA, while potassium acetate cased all other cellular debris and remaining solution to precipitate. Plasmid DNA was the only remaining intact material. The fourth step was to remove all materials not needed in the solution. Plasmid DNA was kept within the tube. Centriguing the solution for 10 minutes at high speed created a separation allowing the top layer to be extracted through pipetting. This top layer consisted purely of Plasmid DNA, where it was transferred to a new experimental tube. Steps six and seven happened concurrently, by precipitating any excess proteins, forcing the DNA into a denser concentration. This was accomplished by mixing the DNA with 300 microliters of isopropanol, causing quick precipitation of the DNA (around ten minutes). Additional mixing using the centrifuge prevented precipitation by the protein material, leaving it suspended, and ready for extraction. Protein material was removed, and a small pellet of DNA was left to air dry for roughly fifteen minutes. The final step, number eight, was to add 20 microliters of TE, consisting of 10 mM Tris.Cl pH 7.5, 1 mM EDTA pH 7.5, and .5 micrograms/millimeters of RNAase A. This mixtures purpose was to secure the DNA plasmid and prevent degradation.
After the DNA was extracted from E. Coli, the DNA was implemented into lab seven for restriction enzyme digestion and PCR in lab eight. Agarose gel electropheris was utilized to determine the efficiency of the procedures.
To prepare the agarose gel for both restuction enzyme and PCR, there must be a well comb placed into a gel filled casting tray. A tight fit must be ensured so that gel will not leak out and allow the creation of even rows and wells for solutions to be added into. To create the gel, one must take 0.5 g of agarose and 50 millimeters of 1XTBE and mix into a 250 millimeter Erlenmeyer flask. The mixed solution must then be heated for one minute in a microwave oven. After the gel melted, 5 microliters of Ethidium Bromide, a fluorescence detector, was added to bind and detect nucleic acids. After cooling to roughly to room temperature, the gel was poured into the gel tray. Final solutions of the restruction enzyme digestion and PCR were added to separate trays, which were then attached to electrical leads that produced 100 volts across the gel. After roughly two hours the gels were placed beneath specific light wavelengths that revealed illuminations in the fluorescent dyes. A picture was taken so distances of the fragments could be measured.
In Lab seven restriction enzyme digestion was performed. The DNA, pGEM-HIS1, from lab six was used to be cut by the restriction enzymes PstI and Pvull. Four steps were used in this process to cut the DNA with restriction enzymes. The first was to label three microcentrifuge tubes with one’s initials and reactions numbers 1, 2, and 3. The second step involved the addition of reagents indicated in Table 1 to the specified tubes. Each tube’s total volume was about 20 microliters. The third step was to incubated the tubes in 37 degrees Celsius to ensure that the enzymes activity will stop within a few hours of incubation. The fourth step was determing the final size of the products through agarose gel electrohpresis. 5 microliters of 6X loading buffer was added to each tube to prepare the products for electrophoresis. Finally, 15 microliters of DNA digest/buffer mix was placed in the lanes created by the well-comb. The movement of the individual products for each lane was then measured to determine the size of the fragments.
In lab eight, PCR’s purpose was to amplify the amipicillin resistant gene in the plasmid pGEM-HIS1. Seven steps were involved this process. The first was to label three 0.2 millimeter tubes with initials and tube numbers 1, 2, and 3. The second was to mix 1 microliter of product with 24 microliters of water in a separate tube. This solution acted as the template for the PCR reactions. The third step was the adding of reagents to the tubes show below in Table 2. Additionally, master mixes 1 and 2 are shown in Tables 3 and 4.
Steps four, five and six were to place the tubes into the thermocycler. The solutions go under this process when placed in the Thermocycler. The initial denaturation step; the solution was heated to 94 degrees to 96 degrees from one to nine minutes, causing the DNA to denature into two linear strands. Denaturation cycle is next, where the reaction is held at the same temperature for an additional minute, that is then followed by the annealing step where the temperature is lowered to 50 to 65 degrees for another minute. This allows the oligonucleotide primers to anneal to the single stranded DNA, where the DNA polymerase will eventually begin to synthesize DNA. The next step, Elongation, uses Taq polyermase, to create a new complmentary strand of the template DNA. Last, final elongation involved the reaction being kept between 70 to 75 degrees for 5 minutes, allowing the DNA to fully extend. The reaction was then kept at 4 degrees until the sample was removed. Agarsoe gel electrophoresis was used to then check if the fragment was adequately amplified.