Bacterial Transformation Lab

Lab Report (Scientific Paper) 2:
Bacterial Transformation;DNA Extraction Part I & II:Total Genomic Extraction & Plasmid Extraction;Electrophoresis

By:Chris Foster

Abstract: We conducted three experiments that included a Bacterial Transformation, a two process DNA extraction, and a final procedure using gel electrophoresis. The Bacterial Transformation lab was performed to prepare the plasmid into a bacteria and to use that bacteria to amplify the plasmid in order to make large quantities of it. This is based on the natural function of a plasmid to transfer genetic information vital to the survival of the bacteria. The next process of DNA extraction involved the extraction of total genomic DNA, and extraction of bacterial plasmids.
The extraction of DNA is important because it is necessary to separate the DNA from other materials so that we can use the DNA for the final electrophoresis process.
The gel electrophoresis portion of the lab is used to separate the DNA by size or charge. DNA Gel electrophoresis is performed for analytical purposes of the bacterial DNA used throughout this experiment.

Introduction: In a series of three lab experiments, we involved three separate linked experiments that started with transforming E.coli bacteria with a special plasmid called the pGLO (+ &-). This is a specialized plasmid that contains an abnormal promoter that is linked to GFP gene from the Aequorea victoria, a jellyfish that contains a transgenic plasmid containing DNA from two.different organisms. We want to speed up the natural process of bacterial transformation weakening the cell membranes of the bacteria through the transformation, transduction, and conjugation of the E.coli. Following this first process of the three step experiment we will being doing the first DNA extraction (Total Genomic Extraction) which involves the goal of separating the DNA from other materials so that they are readily available for further research. We will extract the DNA from our pGLO plasmid (+/-) to be used later on in our experiment
for gel electrophoresis. The second DNA extraction process involves the extraction of the bacterial plasmids after purifying the samples from the previous procedure. A similar process related to the previous extraction is going to be performed to remove the plasmid except using a method that is more efficient. This plasmid DNA will also be used in the next process of gel electrophoresis. The final process of DNA gel electrophoresis involves both the total genomic DNA, and the plasmid DNA that was extracted from the +pGLO and -pGLO used in the first process during the transformation experiment. Using two different dyes, we will prep the the samples with these dyes to accomplish two things. The first dye called bromophenol blue, is used to make the loading process when the samples are ready to injected into the gel easier. The second dye called Ethidium Bromide, a hazardous mutagen and carcinogen used to make the samples in the gel more visible for observation and analysis. The purpose of this is to separate the different separate a mixed population of DNA fragments by length, to estimate the size of DNA fragments.

Materials & Methods: In the first process of this experiment of Bacterial Transformation, began with the use of both a negative and positive pGLO. 1 ml of E.coli was added to the +pGLO then pelleted through centrifuging then 500 micro liters of Ca Cl transformation solution was added to the +pGLO tube and re suspended using pipettiing. 250 micro liters of the transformation solution was added to -pGLO tube then both tubes were placed on ice for 30 min. After, 5 micro liters of of suspended plasmid DNA is added to the +pGLO plasmid only. The tubes are placed back on the ice for 10 more mins. A heat shock is used next inside a 42 degree Celsius hot water bath for exactly 50 seconds then moved back to the ice to cool. 250 micro liters of LB nutrient broth is added to each table then incubated for 5 minuets at room temperature. Next the tubes were transferred to the 37 degree Celsius incubator for 30 mins. A spreading rod is used to spread the bacteria onto the four plates using 100 micoliters. The plates sat for ten minuets so that the solution can get absorbed into the agar. Taped then flipped upside down and stored into the 37 degree Celsius incubator to be stored for the next process. The plates were observed under UV light 24 hours later to see the colony count for observation and analysis.
The next process of the experiment involves a two step procedure of DNA extraction involving extraction of the total genomic DNA and the extraction of the bacterial plasmids (+/-). The first process of extracting the total genomic DNA involved a cell lysis. 1 ml of the overnight culture was added to a 1.5 ml microfuge tube and then centrifuged at exactly 13000 for 2 mins to pellet the cell. 600 micro-liters of Nuclei Lysis solution was added then re suspended by pipetting. Incubation at 80 degrees Celsius was done to lyse the cells then cooled to room temperature. The second process involved a RNase treatment that used 3 micro-liters of Rnase A solution that was added to the cell lysate. The sampler was then mixed invertedly 5 times then incubated at 37 degrees Celsius for 15 minuets. The third process of the experiment involved Protein Precipitation. The Rnase treated sample was cooled to room temperature and then 200 micro-liters of Protein Precipitaion Solution was added to the cell lysate. After shaking the sample for 20 seconds mixing the solution, it was iced for 5 minuets. Then the solution was centrifuged at 13000 for 3 minuets to form a pellet of cellular debris. This prepared us for the fourth process of DNA precipitation. 600 micro-liters of 100% disproportional was added to a new 1.5 microfuge tube. The supernatant liquid containing the DNA was placed into the 1.5 microfuge tube containing the isoproponal. We mixed the solution sample by inverting it until visible fine strands were present the centrifuged the solution at 13000 for 2 min until visible in a pellet. The residual supernatant was poured out and then dumped onto a paper towel then 600 micro-liters of 70% ethanol was added and carefully inverted to wash the DNA pellet then centrifuged again at 13000 for 2 minuets. The ethanol was carefully removed from the solution to preserve the pellet then drained again. The final process involved DNA Hydration that required 50 micro-liters of DNA dehydration solution to be added to the pellet. The the sample was incubated at 65 degrees Celsius for 1 hour to accelerate hydration, shaken 5 times, and then briefly centrifuged to collect sample at the bottom to be stored at 4 degrees Celsius for the next procedure.
The second part of the DNA extraction of the bacterial plasmid involved a prep period adapted by the Bio-Rad Quantum prep plasmid mini-prep kit. 1 ml of overnight culture of plasmid-containing cells was transferred into a micro-centrifuge tube then pelleted by centrifugation for 30 seconds removing any supernatant. The next two steps were timed at 10 minuets slowly and gently. Adding 200 micro-liters of the Cell Re-suspension Solution then re-suspending the pellet was done first. Next, 250 micro-liters of the Cell Lysis Solution was mixed in and inverted 10 times to create a viscous and slightly clear solution. 250 micro-liters of Neutralization Solution was mixed in and inverted 10 times to form a visible precipitate. Centrifuging for 5 minuets to form a compact white pellet was done while also preparing two wash tubes with spin filters . The Quantum Prep Matrix was mixed by shaking repeatedly until re suspended The supernatant from the last step is added to the spin filter along with 200 micro-liters of suspended matrix. Then the sample was centrifuged for 30 seconds and the spin filter is removed and the filtrate at the bottom of the tube is discarded. 500 micro-liters of Wash Buffer is added to the sample then washed by centrifuging. The spin filter is to be removed and then the filtrate is to be discarded once again. 500 micro-liters of Wash Buffer is added for another wash of the matrix and then centrifuged for two minuets to remove the traces of Ethanol. The spin filter is then removed and the tube is discarded keeping the spin filter. The filter is then placed onto one of the 1.5 ml collection tubes. Finally, 50 micro-liters of warm distilled H20 is added then centrifuged to eluted the DNA for one minute. The spin filter is discarded and the eluted DNA is stored at -20 degrees Celsius for the next step in this experiment.
The final portion of this experiment involves the use of DNA electrophoresis on both the total genomic DNA, and the plasmid DNA extracted from the +pGLO plasmid and the -pGLO plasmid bacteria used earlier in the transformation experiment. A gel mold is prepared and a comb is inserted into the end slots of the mold. Then 30ml of diluted TBE buffer of 1X is added to the .18 grams of agarose in a Erlenmeyer flask to make a 0.7% w/v gel. The solution is heated until boiling in a microwave, the cooled in a flask. 3 micro-liters of Ethidium Bromide solution is added to the mix then poured into the gel mold. After sitting for 20 mins, the gel is solidified then the electrophoresis tray is filled with buffer and the extracts a mixed well. 10 micro-liters of each extract is separated into two microfuge tubes. 5 micro-liters of a DNA ladder is added to a new 0.5 microfuge tube. 2.2 micro-liters of the 6X loading dye is added to each sample. After the gel is loaded onto the buffer tray, the combs are removed from the gel and the transfer of all prepared DNA extract is loaded into the wells of the gel. The power supply is turned on and set to 200 volts to run for 30 mins or until the gels are 0.5 cm from the end of the gel. The gel is then taken out and analyzed under UV light for observation of the distance of the bands and then photographed for documentation.

Results: In the Bacterial Transformation experiment, two plates were were inoculated with untransformed (-DNA) bacteria. \ The other two plates inoculated with transformed (+DNA) bacteria. Bacteria that grow on these antibiotic-containing plates have been transformed. They picked plasmid DNA that contains a gene that gives them resistance to ampicillin. After the extraction of both the total genomic DNA, and the bacterial plasmids, the DNA was analyzed during electrophoresis. Unfortunately, none of the samples were visible during UV observation causing our results to be of no use, only the ladders showed up and we could not measure the lengths of the gels and sizes.

Discussion: Although the experiment went very well for mostly the whole procedure, many steps in our experiments were done poorly which resulted in the absence of results from the final process of the experiment.
In the Bacterial Transformation portion of the lab, we successfully weakened the bacterial cell membranes of the plasmids. In an article by Dipshikha Chakravortty, a similar bacterial transformation experiment was done using controlled micro-shockwaes to develop a unique bacterial transformation. The conditions were optimized for the maximum transformation efficiency in E.coli. This experiment was similar to the one we performed in that each experiment successfully proved that transformation can be sped up and achieved through various methods . Wanshen Yang wrote an article that uses “A multifunctional magnetic nanoparticle (MNP)-assisted bio separation method was developed to isolate plasmid DNA(pDNA) from Escherichia coli culture. Using the pH-sensitive carboxyl-modified magnetic nanoparticles, both cell capture and the subsequent removal of genomic DNA/protein complex after lysis can be achieved simply by magnetic separation.” This method is cost effective, and does not require centrifugation or precipitation steps and has the potential for automated DNA extraction. The process we used was not as efficient as this method but more cost effective in obtaining the same results. The gel electrophoresis portion of this lab was the worst of all three portions, due to
the mistakes we made during the processes of the lab we did not obtain any results causing a fail of the experiment.

Dipshikha Chakravortty, et al. "Bacterial Transformation Using Micro-Shock Waves." Analytical Biochemistry 419.2 (2011): 292-301.Academic Search Complete. Web. 23 Apr. 2012.
Wanshen Yang, et al. "Bacteria Capture, Lysate Clearance, And Plasmid DNA Extraction Using Ph-Sensitive Multifunctional Magnetic Nanoparticles." Analytical Biochemistry 398.1 (2010): 120-122. Academic Search Complete. Web. 23 Apr. 2012.
P. Fach, et al. "Real-Time PCR And Enzyme-Linked Fluorescent Assay Methods For Detecting Shiga-Toxin-Producing Escherichia Coli In Mincemeat Samples." Canadian Journal Of Microbiology 53.3 (2007): 337-342. Academic Search Complete. Web. 23 Apr. 2012.