Transfection Lab

coli the bacteria that will be used to house the GFP is the competent cell. They are called competent cells because they are able to accept foreign DNA or plasmids. They are first observed in 1928 by Frederick Griffith where he proposed that bacteria are capable transferring genetic information (Havarstein, 2010). These cells such as E. coli are normally made competent by exposure to a rich calcium environment, where the mixture calcium chloride will be used in. Calcium chloride’s positive charges will cancel out the negative charge of the plasmid and E. coli’s cell wall weakening it, making it easy to pass.

In order to find transformation efficiency, the total number of colonies on the plate is divided by the total amount (µg) of DNA spread on the plate. All the factors that must be taken into account while finding the transformation efficiency include; the total amount (µg) of plasmid DNA used, the total volume (µl) of cell suspension prepared, the fraction of DNA spread on the plate and finally the total amount (µg) of DNA present on the plate. In the LB/AMPC plate the transformation efficiency was 8.2 x 103 colonies per µg of plasmid DNA while the LB/AMPlux plate the transformation efficiency was 1.07 x 104 colonies per µg of plasmid DNA. The transformation efficiency for the LBC and LBlux plates were not taken into account, even though they too transformed because they were not in a restrictive environment where they needed to express their ampicillin resistance genes in order to

The product that will result by genetically modifying this organism’s genome is known as a recombinant protein. The process will combine the human protein with a vector, a specific DNA molecule, which will transport the genetic material. This procedure will use a bacterial plasmid. The plasmid is

After the incubation, 1.5 mL of each of the three cultures were added to eppendorf tubes and centrifuged at 13,200 rpm for 1 minute. An alkaline lysis procedure like that of Birnboim and Doly was then performed to extract the plasmid DNA with 200 μl of alkaline SDS detergent solution (Birnboim & Doly, 1979). After

Another improvement that can aid in the accuracy of the experiment would be to use combinational cloning, a less complex and involved technique for efficient cloning of different vectors with various clone sites (Zhang and Tandon, 2013). The technique uses originally existing clone sites from circular plasmids to prepare the inserts to achieve maximal correct digestion of the inserts (Zhang and Tandon,

The data indicate that the E. coli bacterium is a poor choice for DNA transfections, at least with this specific culture media.

typhimurium was ran on the first day to determine the CFU/mL of the overnight culture in order to calculate the volume of phage needed for transduction, the dilution of 10-8 was used since 93 colonies (9.3 x 109 CFU/mL) grew on the LB plate (See table 1). A titration of P22 phage and S. typhimurium were also performed to determine the PFU/mL of the P22 phage, a control plate with phage alone showed now growth, and the dilution 10-7 was found to have 132 plaques (1.32 x 109 PFU/mL). This value was used together with the dilution results of S. typhimurium on day 1 for calculating the volume of phage (see sample calculation). An MOI of 0.01 was used with these two values (volume of bacteria was .1mL) in order to determine the volume of phage needed for transduction, which ended up being 7µl. Using this information, three plates were run for the transduction, one plate with cells only on LB-CM, a plate with the cells and the phage on LB-CM, and a plate with phage only on LB-CM. There was only growth on the cells and phage plate, giving 158 colonies (1.58x103/mL) (see figure 1. And table 1. for results). This indicated successful transduction of the chloramphenicol gene since only colonies with the phage were able to grow on the antibiotic plate. A “final” titration was simultaneously run for the specific sample of recipient S. typhimurium actually used in the transduction, with the dilution of 10-8 resulting in 262 colonies (2.62 x 1010

In genetic transformation, vectors are used to transfer genes from organism to another. In this experiment, we used plasmids as

Numerous standard laboratory techniques and instrumentation was used to obtain the forthcoming results. The ASAP1 expression plasmids were created by standard molecular biology methods, and then verified through cloned fragment sequencing. A plasmid was used for multiple reasons, the most prominent being that plasmids are very easy to manipulate to express a desired genetic element. In addition, they are very stable as well as self-replicating once initially constructed. HEK293A cells were used in experimentation and transfected with the ASAP1 plasmid. The HEK293A cells, which are very popular for transfection, were chosen as they are easy to reproduce, maintain, and have a high efficiency of transfection. HEK293A cells also have minimal processes that need to be accomplished in order to preform voltage-clamp experimentation. Transfection is simply the process by which

In order to make recombinant DNA the insert must be inserted into the plasmid. To do this, more insert than vector should be added to a new tube and to a ligation kit the following should be added: 2µl of warm vortexed buffer 2, 10µl warm vortexed buffer 1 and 1µl of buffer 3. After this the solution should be incubated at room temp for 15 minutes and then the transformation step should be carried out as below.

Use the ultrasound to enhance cell permeability is a new methods for gene transfection. The increase uptake of DNA or RNA has been demonstrated by several investigators2-1, 2-5. However, the further enhancement of gene transfection

We labeled the two microcentrifuge tubes, one “+pGLO” and the other “-pGLO”, then put on our gloves. We pipetted the 500 µl of transformation solution into these two tubes, half (250 µl) in each, and put these tubes on ice. Then we put a colony of E. Coli bacteria into each tube and put pGLO plasmid DNA into the +pGLO tube, using sterile loops. We put the tubes back on ice, timing it for 10 minutes. During the interim, we retrieved and labeled the bottom of each of the four LB nutrient agar plates, according to what they contained and would contain. We then took both tubes out of the ice and immediately placed them into a floating rack

Then, we placed 100 µl of the E. coli cells into four Eppendorf tubes each and labelled them A, B, C and D. After filling them up, we placed them into an ice box. After this, we needed to perform the transformation process by mixing the E. coli cells with different concentrations of plasmid DNA. In test tube A, we added 5 µl of distilled water to the E. coli cells. In test tube B, we added 2.5 µl of undiluted plasmid DNA to the E. coli cells. In test tube C, we added 1.0 µl of undiluted plasmid DNA to the E. coli cells. Lastly, in test tube D, we added 1.0 µl of plasmid DNA diluted 1 in 10 to the E. coli cells. Then, we used a P100 pipette and mixed all the liquids in all the Eppendorf tubes by pipetting the solution up and down. Then, we left the Eppendorf tubes along with its contents in the ice box for twenty minutes. After twenty minutes, we labelled the base of the four agar plates to match with the appropriate plasmid DNA concentration about to be plated. We also wrote our initials, the date, and the table number on the base of the agar plate. We then gently lifted up the cover of the agar plate with just enough space to allow us to plate each tube of cells onto four different agar plates – one agar plate to one plasmid DNA concentration. We used a spreader to spread the cells evenly over the surface of the agar, being careful not let the spreader touch

In this experiment, individuals were able to use recombinant DNA techniques such as cloning to alter the DNA of a certain strain of yeast called Saccharomyces Cerevisiae. By the use of cloning, scientists are able to analyze and compare the genetic material of a specific organism to another. In this lab, the recombinant DNA techniques are used to transform AmpS E. Coli to AmpR E. Coli. This was done by using an insert, a vector, and a host. The insert was S. Cerevisiae genomic DNA, E. Coli plasmid was the vector, and E. Coli was the host. The technique of gel electrophoresis was also used to determine whether the DNA was cut or not with restriction enzymes. Uncut sequences of the DNA were sent to an outside laboratory for sequencing. Then when the sequencing was complete, computer programs, such as BLAST, were used to determine whether or not the genomic insert originates from a gene and if the yeast codes for a protein that could be found within the human genome.

Transfection and selection was pursuant to previous description [40]. The empty vector served as the negative control. For the EGFP-LC3 cell line, the pEGFP-LC3 overexpression plasmid was previously prepared in our lab and used for cell transfection, followed by G418 (100 μg/ml) selection.