Plasmid Identification Project April 30, 2014 Plasmid Identification Project Biotech 1015 Whitney Morris April 30, 2014 Plasmid Identification Project April 30, 2014 Introduction What is a plasmid? A plasmid is a circular piece of DNA that is smaller than a chromosome and is used to carry genes into a cell. Plasmids are very useful in finding and isolating a gene, because they replicate independently. When identifying a plasmid a restriction enzyme is used. A restriction enzyme is an enzyme that cuts DNA into a specific sequence. You will then run the plasmid and enzyme through a gel electrophoresis. Gel electrophoresis will separate the DNA, of the plasmid, using an agarose gel. This separation is based on size, the DNA runs from the black electrode (negative) to the red electrode (positive). Understanding the information explained above will allow you to understand the basis of this experiment. The goal of this experiment was to identify an unknown plasmid by using three restriction enzymes and then running a gel to help identify the unknown plasmid. Identifying the plasmid demonstrates the skills learned in Biotech 1015. The strategy of my experiment was to use the restriction enzymes BamHI, PstI and Pvul to cut and identify my unknown plasmid. By using those restriction enzymes, which run in buffer three, allowed me to go through a process of elimination which permitted me to identify the plasmid. Methods The code for my unknown plasmid was: 7936A65. For this assignment I was given the concentration of my plasmid due to the limit on time.. The concentration of my plasmid was, 150ng/ 1µL, which then allowed me to find the amount of unknown plasmid, dH2O, each enzyme, and buffer 3.The amounts can be found in the appendix in 2 Plasmid Identification Project April 30, 2014 table 1. The DNA latter used was a NEB1Kb from the New England Bio labs, as well as the restriction enzymes and buffers. The digest recipes I used were a single digest with BamHI, and a double digest with PstI + Pvul. My sample incubated for one hour at 37 degrees Celsius in a hot plate. The gel I made was a 1% agarose gel in 50 mL of 1x TAE. I had to diluted a 10x TAE to a 1x TAE by adding 5 mL of the 10x TAE to 45 mL of dH2O. I used 0.5 grams of agarose and added that to the beaker containing the 50 mL of the dilute 1x TAE. I then heated the solution in the microwave for four 30-second intervals, until the solution became clear. After heating the solution I added 5 µL of ethidium bromide to the solution. Once the solution had cooled enough I poured the solution into the gel box and allowed it to sit for 30 minutes. Once my sample had finished incubating I added 4 µL of 6x loading dye to the solution. The loading dye allows the sample to be seen as it runs through the gel. I then inserted the 1 KB ladder into the gel on both sides of the gel. I then inserted each sample into a well. I ran the gel at 140 volts until the bottom dye band was between the four and five, which took roughly one hour. The buffer used in the gel box was 280 mL of a 1X TAE. After my gel was finished running I took a picture of it on the UV imaging system, which can be seen in table 2. I was able to determine the sizes of my DNA from the latter as well as from data collected from the NEB cutter website, which provided the sizes of the bands that the enzyme would cut. By using the NEB cutter website, I was able to get an idea of what each gel would generally look like depending on where the sample was cut in the gel. I then made a standard curve graph based on the distance from the middle of the wells to 3 Plasmid Identification Project April 30, 2014 the middle of the band. The standard curve allowed me to gain a greater understanding on the values of the bands in the gel. Results and Conclusion The concentration of my DNA was 150 ng/ 1 µL, I needed 2 µL of 10X NEBbuffer 3, the total volume of the pCR tube needed to be 20 µL. With each of my digests and the control, they contained either 1 µL of the enzymes needed in that digests or none at all. The PCR tube had 1 µL or no enzymes, which allowed me to find the remaining amount of dH2O needed for the pCR tube. The data for the amount shown to the side is the gel lists the DNA Latter sizes. The latter was run in the first well on the left and the second to last well on the right. The third well from the left was my control sample, fifth one in, was the sample containing BamHI, and the seventh one in contained PstI + Pvul. By creating a standard curve, which is shown in the appendix labeled table 3, it allows a general idea of where the cuts on the DNA should be located. Listed below table 3 in the appendix, in the table are all of the possible base pair sizes, which would be on the cut DNA for the plasmids. By looking at those cut sizes and where they are located on the gel, I was able to determine which plasmid I was given. Based off of the information and from the data collected I was able to come to the conclusion that my plasmid was pBLU. I was able to come to this conclusion because when I compared my gel to where the cuts on the other plasmids the pBLU was the only possible reason for a cut above the ladder, roughly at 6046. My sample wasn’t incubated enough so the DNA wasn’t cut completely causing the DNA to be above the ladder. 4 Plasmid Identification Project April 30, 2014 When attempting a second test on my unknown plasmid, my samples were not incubated at the right temperature for the right amount of time. Figure 2 shows that none of the DNA was digested, so none of the DNA went farther down then the ladder. Due to the lack in data from the gel, I was not able to draw any conclusions from the gel or create a standard curve from the gel. Based on the results from my first test, pBLU is able to account for all of the cuts in the PstI + Pvul test; pAMP and pKAN could only match two bands and still had a distance close to 150 between the actual band and where it was supposed to cut based on the data. Looking at all the data and by using the R-value off my Standard Curve I concluded that my plasmid is pBLU. 5 Plasmid Identification Project April 30, 2014 Appendix Table 1: Tube Unknown 2 µL Control 2 µL BamHI PstI + Pvul 2 µL 10x NEB Buffer 2 µL 2 µL 2 µL dH2O BamHI PstI Pvul 16 µL 15 µL 14 µL ---------1 µL ---------- ---------- ----------------- --------1 µL 1 µL Total volume 20 µL 20 µL 20 µL Figure 1 First row on left: 1KB Ladder Size of bands: 10, 8, 6, 5, 4, 3, 2, 1.5, 1, 0.5 Second row: Control Third row: BamHI Fourth row: PstI + Pvul Fifth row: KB Ladder Size of bands: 10, 8, 6, 5, 4, 3, 2, 1.5, 1, 0.5 6 Plasmid Identification Project April 30, 2014 Figure 2 First row on left: 1KB Ladder Size of bands: 10, 8, 6, 5, 4, 3, 2, 1.5, 1, 0.5 Second row: Control Third row: PstI+ Pvul Forth row: BglI Fifth row: BamHI Sixth row: HindIII Seventh row: 1KB Ladder Size of bands: 10, 8, 6, 5, 4, 3, 2, 1.5, 1, 0.5 Standard Curve Band Migration (mm) 12000 10000 y = 6E+07x-2.859 R² = 0.99966 8000 6000 4000 2000 0 0 10 20 30 40 50 Ladder Size (bp) 7 Plasmid Identification Project April 30, 2014 Band Migration (mm) Ladder - Band 1 Ladder - Band 2 Ladder - Band 3 Ladder - Band 4 Ladder - Band 5 Ladder - Band 6 Ladder - Band 7 Ladder - Band 8 Ladder - Band 9 Control - Band 1 BamHI - Band 1 BamHI - Band 2 BamHI - Band 3 PstI+PvuI - Band 1 PstI+PvuI - Band 2 PstI+PvuI - Band 3 PstI+PvuI - Band 4 PstI+PvuI - Band 5 PstI+PvuI - Band 6 Ladder Size (bp) 21 23 25.2 27 29.2 32 37 40.8 47.5 12.2 12.2 19 25 28.5 35 39 42.6 46.4 50.5 10000 8000 6000 5000 4000 3000 2000 1500 1000 47016 47016 13249 6046 4157 2310 1695 1317 1032 810 Table 4: Base pair cuts # of cuts 1 Enzymes BamHI-BamHI 1 2 3 PvuI-PstI PstI-PvuI Pvul-Pvul Circular Sequence: pAMP Coordinates 1018-1017 3157-946 947-2260 2261-3156 Length (bp) 4539 2329 1314 896 Circular Sequence: pKAN # of cuts Enzymes Coordinates Length (bp) 1 BamHI-BamHI 2096-2095 4194 1 2 3 4 Pvul-PstI PstI-PstI Pvul-Pvul PstI-Pvul 2812-771 772-1694 2240-2811 1695-2239 2154 923 572 545 8 Plasmid Identification Project April 30, 2014 # of cuts 1 1 2 3 4 5 6 7 8 Circular Sequence: pBLU Coordinates Enzymes BamHI-BamHI 3161-3160 Pvul-Pvul PstI-PstI Pvul-Pvul Pvul-Pvul Pvul-Pvul PstI-PstI Pvul-PstI PstI- Pvul 4821-1363 3380-4695 2297-3022 1817-2296 1364-1816 3183-3379 3023-3182 4696-4820 Length (bp) 5437 1980 1316 726 480 453 197 160 125 Bibliography BioLabs, N. E. (2003). Double Digest Finder . Retrieved April 30, 2014, from New England BioLabs : https://www.neb.com/tools-and-resources/interactivetools/double-digest-finder BioLabs, N. E. (2003). NEBuffer Performance Chart. Retrieved April 30, 2014, from New England BioLabs: https://www.neb.com/~/media/NebUs/Files/nebufferperformance-chart-with-restriction-enzymes.pdf Laboritory, C. S. (n.d.). DNA Learning Center . Retrieved April 30, 2014, from Cold Spring Harbor Laboratory : http://www.dnalc.org/resources/plasmids.html Vincze, T. P. (2003). BioLabs . Retrieved April 30, 2014, from NEBcutter: http://tools.neb.com/NEBcutter2/index.php 9