Plasmid DNA is a circular form of self-replicating DNA that scientists can manipulate to carry and clone other pieces of DNA Thieman Palladino 2009 strands are found in the cytoplasm of a bacterium that scientists can maneuver to conduct and clone other parts of DNA Plasmids is found in cells which the cells are then used as vectors because of their outstanding capability to transport genes that allow bacteria to grow in contrary circumstances Plasmids wouldn't be able to take in and break down without the help of restriction enzymes The goal for the enzymes is to cut the DNA at a specified sequence of nucleotides The goal here is to clear any DNA molecules that was not needed at the time of growth To determine the size fragments from how the restriction enzymes cut a tool called the Gel Electrophoresis was used Gel electrophoresis is one of the most accepted ways to figure out the DNA sequences that researchers have from DNA This skill will take assistance of the negative charge on DNA molecules by applying the electrical field which will implement the energy to abstract the DNA molecules proportioned to the size The goal of this experiment is to figure out the unknown plasmid that the instructor has given to help in the discovery of the unknown plasmid and there were three to figure out from pAMP pKAN or pBLU
This goal is important to my understanding of biotechnology lab techniques because it is through the use of the lab that rules and procedures of the lab are learned For example this is a lab where we need to be dressed appropriately for the use ethidium bromide involved which could cause the individuals who are experimenting to have his her DNA mutate Also knowing the math involved would be essential in knowing how much to put buffer with the specific restriction enzyme The plasmid and the dH20 in one tube The steady hands in pipetting is also very crucial for this lab because it could alter up your calculations Strategies that I used to achieve this goal was to go through and review more from the past labs that the class has done This final lab is to test us whether or not we have mastered the concepts taught in this biotechnology course Methods The unknown plasmid code name 1353E94 For the final concentration calculation our instructor gave the concentration of our plasmid which was 1 96 ug uL The source of the DNA marker or DNA ladder we were able to use was DNA 1Kb ladder from tinyurl com nebdoubledigest and tinyurl com NERcutter NEB The sources of enzymes that I used were Pstl Ncol Sacl and Kpnl The information from these restriction enzymes were all available from the NEB website Shown below is the table of the percent activity in the specific NE Buffers with specific enzyme The digest recipe that was used digests one double digest The double digest recipe we used were Ncol HF and Kpnl HF For our single digest it was Nacl HF and also one uncut control of the unknown plasmid
The four finished tubes that were put in the incubator for twenty four hours and set it to 37 celsius After we incubated the tubes 1 agarose gel was made at a volume of 50mL with 0 8 grams of agarose powder and 40mL of 1xTAE buffer into a flask Then we microwaved it for about a minute till the whole thing was melted Next we needed to add the 1 ul of ethidium bromide The agarose was poured onto the gel tray and was cooled The gel was finished and ready to run after the gels were inserted Prior to the experiment the agarose liquid was poured in the gel tray the sanitized 10 well comb was put into the gel tray During the time that the gel was hardening I filled the four tubes with the exact amount of 7uL of SYBR Green Dye A DNA ladder was obtained from the instructor after the gel hardened I pulled out the 10 well comb and poured the 1xTAE buffer to cover the gel and the wells Then I fixed the four tubes including the 1Kb ladder with 22uL from each tube into their separate wells 1 2 3 4 5 Figure 2 shows the order which each enzyme tube was placed in the well The gel was run at 120 volts for one hour at which point the lines were visible within one inch from the bottom of the gel After the gel ran for about an hour the gel was then taken to the UV Transilluminator to record the image Figure 3 The UV Transilluminator image of the gel To make a firm decision of the size of the DNA fragments from the gel is to compare the ladder fragments kbp measurements Also fragments can be determined by measuring the fragments of the ladder in millimeters and put the information onto an excel sheet and make a scatter plot and a standard curve to assist in determining the correct size for the other fragments By using the website http tools neb com NEBcutter2 I could digest all four plasmids with restriction enzymes that I have chosen I can also view what they gel may have looked like for each of the plasmids
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