Pb325 Plasmid

In this case, our plasmid DNA is isolated from a liquid culture of the E.coli that was transformed. This is done by reacting the liquid culture with five buffers. The buffers are P1, P2, N3, PE, and an elution buffer. P1 is used to re-suspend the pellet and degrade RNA, P2 is used to lyse cell membrane, PE is used to wash the sample, the elution buffer is used to release DNA from the spin column, and N3 is used to precipitate proteins and genomic DNA. The main components of P1 are Tris, EDTA, and RNase. The main components of P2 are NaOH and SDS. The main component of PE is ethanol and the main component of N3 is acetic acid. The main component elution buffer is water. The possible contaminations of mini-prep are proteins and salts (Garey et al.,

E. coli HB101 was transformed with pGLO plasmid then grown on media containing ampicillin and/or arabinose and on medium containing neither (Brown, 2011). This is done for selection of transformed cells since not all cells are expected to take up the plasmid (Brown, 2011). We also expect roughly the same CFU on any plate(s) receiving samples from the same microcentrifuge tube, since they are getting the exact same

First, 50 uL of bacterial cells were made competent by being treated with 590 uL of CaCl2 in three different test tubes before transformation of the plasmids took place. In order to enhance the uptake of the plasmid DNA the instructor added the CaCl2 solution to the bacterial cells. The tubes were placed into a cold ice bath for about ten minutes after the CaCl2 solution was added into the test tubes of E. Coli.

In this investigation pUC19 plasmids were used as the vector due to its small size of 2686bp, high uptake efficiency by the host and fast replication time. Important features of this plasmid include the origin of replication and multiple cloning sites (MCS). The origin of replication allows the plasmid to replicate inside the host bacterium. The MCS is located within the lacZ gene and contains unique sites for the Xbal & EcoRI restirction enzymes to cut and produce sticky ends for the CIH-1 gene to bind to. Furthermore, the pUC19 plasmid also contains an ampiccilin resistance gene so only transforemed E.coli are able to remain viable when spread on the agar plates that also has the addition of ampiccilin. The lacZ gene encodes the β-galactosidase enzyme which aids in indentifying the recombinant E.coli from the non recombinant cells (Coventry University 2016).

70µL of competent E.coli are added to both test tubes; pUC18 and Lux (Alberte et al., 2012). Both test tubes are then tapped and placed back into the ice bath for 15 minutes. While waiting, another test tube is obtained, filled with 35µL of competent cells and labeled NP for no plasmid. A water bath is preheated to 37 degrees Celsius and all three labeled test tubes are inserted into the bath for five minutes (Alberte et al., 2012). Using a sterile pipet 300µL of nutrient broth are inserted into both the control and Lux test tubes and 150µL are inserted to the no plasmid test tube to increase bacterial growth. All three test tubes are then incubated at 37 degrees for 45 minutes. Six agar plates are obtained and labeled to correspond each test tube, three of the plates contain ampicillin. A pipet is used to remove 130µl from each test tube containing a plasmid and insert it into the corresponding agar plate. For this, a cell spreader is first

The plasmid pGLO contains an antibiotic-resistance gene, ampR, and the GFP gene is regulated by the control region of the ara operon. Ampicillin is an antibiotic that kills E. coli, so if E. coli, so if E. coli cells contain the ampicillin-resistance gene, the cells can survive exposure to ampicillin since the ampicillin-resistance gene encodes an enzyme that inactivates the antibiotic. Thus, transformed E. coli cells containing ampicillin-resistance plasmids can easily be selected simply growing the bacteria in the presence of ampicillin-only the transformed cells survive. The ara control region regulates GFP expression by the addition of arabinose, so the GFP gene can be turned on and

The purpose of the experiment was to isolate plasmid DNA, followed by restriction digestion using restriction endonucleases and then visualizing the digested fragments after subjecting to gel electrophoresis. Plasmid DNA (pSP72 DNA) was isolated from Escherichia coli KAM32 (E.coli) cultures using the QIA prep miniprep kit and then subjected to restriction digestion by EcoRI and HindIII. The restriction digested DNA was then loaded into the wells of 0.7% agarose gel and subjected to electrophoresis. It can be concluded from our results that our plasmid DNA isolation was successful and the restriction digestion results were partially in agreement with our hypothesis.

The experiment was conducted in two main parts: in the first part, the single and double restriction digests were run to cut the plasmids using combinations of three different enzymes. In the second part, the fragments resulting from each reaction were separated using gel electrophoresis. 1) Restriction Digests In this part of the experiment, seven different samples were prepared with different combination of reactions. Given the unknown plasmid and three restriction enzyme (HindIII, BglI and PvuII), seven different Eppendorf tubes were prepared to account for 3 single digests, one with each of the three restriction enzymes, 3 double digests combining two enzymes at a time and alternating between all the possible combinations, and also for the control reaction where no enzymes were added to the plasmid.

Isolation of plasmid DNA from three cultures of E.coli using a method known as the alkaline lysis method.

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

An alkaline lysis procedure was performed and followed by binding to a silica gel filter. The lysis released plasmid DNA from the cells and the purification on the binding membrane separate the plasmid DNA from bacterial cell suspensions. There is a possible error in this section that can lead to contamination of plasmid DNA with chromosomal DNA. Vigorous stirring can force chromosomal DNA to be in the supernatant along with plasmid DNA. Therefore, mixing during the lysis procedure should be carried out by only gentle inversion. For the second part of this section, the DNA concentration was determined using a nanodrop spectrophotometer. The purity, contamination, and absorbance were also found along with the DNA concentration. All

In experiment of Bacterial plasmid DNA extraction, solution 1 which consists of ice-cold resuspension buffer helped corrupting the cell membrane. Because cell membrane has not been broken down yet the collected blend is clear. However, later on inclusion of Solution 2 which is lysis buffer the cell membrane was broken. The solution 3 that is a neutralization buffer was added and it provided the contaminated genomic DNA to precipitate. Using centrifugation method provided the plasmid DNA to isolate from cell debris and genomic DNA. Protein contamination was tried to prevent using ethanol.

A plasmid vector must have (1) an origin of replication to allow it to be replicated in E. coli independently of the chromosome, and (2) a selectable maker , usually antibiotic resistance. The selectable maker allows the presence of the plasmid to be easily identified through selection, cells that contain the maker will live when plated on antibiotic –containing growth media, and cells that lack the plasmid will not live (they are killed by the antibiotic). A fragment of DNA is inserted by the techniques described in figure 16.2 in a region of the plasmid called the multiple cloning site (MCS).

Bacterial transformation is the process of moving genes from a living thing to another with the help of a plasmid.The plasmid is able to help replicate the chromosomes by themselves; laboratories use these to aid in gene multiplication. Bacterial transformation is relevant in everyday lives due to the fact that almost all plasmids carry a bacterial origin of replication and an antibiotic resistance gene(“Addgene: Protocol - How to Do a Bacterial

Plasmid DNA with Restriction Digest: The purpose of restriction digest of plasmid DNA is to understand how each DNA plasmids was cut with the given restriction enzymes and perform gel electrophoresis to observe the samples. Nine restriction digests were created, containing three digests for each of the three plasmid DNAs identifying as recombinant, non-recombinant, and unknown. Out of the nine digests, six are actual digests and three are undigested controls. A master mix is created to add to each of the nine samples with its following stock ingredients: 10 ul of 2X Reaction Buffer, 1 ul of Nco1, X ul of sterile water (Single digest), 10 ul of 2X Reaction Buffer, 10 ul plasmid DNA, 1 ul Nco1, 1 ul of Not1, and X ul of sterile water (Double