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... ting smaller fragments that travel longer distances than the single cut or uncut, during electrophoresis. Which in turn proves that segment sizes are proportional to distance migrated. On a standard curve, the distance migrated is used in order to extrapolate the size of the DNA (base pairs). For an undigested or uncut p KAN plasmid it is expected that one fragment will be shown, unless the plasmid has been nicked and then will therefore show three. The size of this fragment should be close to 4207 base pairs.
The single digested p KAN plasmid is expected to show one fragment. The size of this fragment being close to 4207 base pairs. Finally, the double digested p KAN is expected to have two fragments, and the sizes of these fragments should be 1875 and 2332, base pairs. As for the transformation experiment it is expected that there will be growth on the LB/KAN if our unknown contains p KAN or there will be growth on the LB/AMP if our unknown contains p AMP. Materials and Methods The first portion of the experiment, bacterial transformation, consists of multiple steps. Because we were performing an artificial transformation of E.
coli DH 5 a, we followed four major steps: pre incubation, incubation, Heat shock, and recovery. The E. coli was pre incubated before lab and therefore was not conducted by us. In incubation we were given five microguge tubes: clear containing TE (no DNA), brown containing p AMP (w/ DNA), purple which was p KAN (DNA), blue containing an unknown. All microfuge tubes contained 5 micrometers of DNA or TE. Using hand pippetor, we transfered 100 micrometers of competent cells to each DNA microfuge tube.
Make sure to replace with a new tip each time . Following that the tubes were incubated on ice for 30 minutes. Heat shock was immediately induced, where the tubes of cells and DNA were placed into 42 degree celsius water bath for exactly 45 seconds, then rapidly transferred back to ice for two minutes. The next step is recovery, where 0. 9 ml of LB broth is added to each microfuge tube, and then incubated in 37 degree celsius water bath for 60 minutes. Lastly the samples were plated. 100 micrometers of the cells and DNA suspension to the center of each agar plate -- LB, LB/AMP, LB/KAN. Using a sterile hockey stick, confluence streaking was executed, and then the plates were incubated for at least 16 hours at 37 degrees celsius.
In the second portion of our experiment, we analyzed DNA. DNA is analyzed by gel electrophoresis. The DNA that we used came from the blue tubes. In this process DNA molecules migrate to the positive pole as current passes through the gel. The common material used for gel electrophoresis of DNA is agarose.
Aragose gel was poured into the buffer tray, which was placed into the gel box. As the arose cools to room temperature, it solidifies to produce the gel with indentations called wells, created by the comb placed into the liquid arose before cooling. A buffer is then added to cover the gel. Then three microfuge tubes (blue) were three different treatments of our unknown plasmid. Which were U, S (cut by Hindi III), and D (Hindi III and then Bam HI). We also recieved a clear tube containing our lambda marker.
Each treatment in the blue tubes, and also the clear tube containing the lambda marker, held 10 micrometers of DNA and to this was added 2 micrometers of loading dye. After the dye was added the tubes were microfuged. Then the DNA (all 12 micrometers within the tubes) is loaded into the wells using a micropipettor. After the wells are filled, the power supply is turned on and the DNA moves towards the positive pole. At this stage the DNA band (s) cannot be seen, but the tracking dye (dark blue) allows the progress of the electrophoresis to be followed. The smallest DNA fragments typically will follow the blue dye down the gel.
The DNA is visualized by staining, with ethidium bromide (Et Br), and then detaining in water. The Et Br binds to the DNA and makes it fluoresce orange under UV light. A photograph of the results is then taken and used for analysis and for a permanent record of our experiment (1). Results Our results from our gel electrophoresis is as follows; in the first well from the left contained out lambda (our positive control), seven fragments were shown, but the last fragment was extremely faint.
The lambda was in the form of linear, and showed the true size of each fragment. The second well from the left was the undigested / uncut (negative control) sample, and showed three fragments rather than one. The reason for this is that it took on the physical form of being a nicked super coil, therefore there were three fragments instead of one. The third well from the left, contained single digested, where one band formed and this is because it took on a linear form.
The fourth well, was the double digested and showed two fragments, because it was cut into two linear peices. In reference to table II, we are able to see the correlation between the distance migrated and the size of visible DNA. As the fragment migrates 8 mm it is found that there are 23, 130 base pairs, 10 mm has 9416, 11 mm has 6557, 13 mm has 4361, 19 mm has 2322, 25 mm has 2027, and 30 mm has 564 base pairs. These numbers are then used to create a standard curve, figure one.
When looking at Figure one, it is seen that as the size of the DNA fragments (base pairs) decreases the distance migrated increases, therefore it can be seen that they are inversely proportional. By using the standard curve it was easy for us to extrapolate the fragment size in the U, S, and D lanes. Treatment U migrated 8 mm and therefore having 23130 base pairs, the second fragment migrated 10 mm which is 9416 base pairs, and the third fragment correlates to 3500 base pairs approximately. Treatment S, the first fragment migrated 9 mm which is 10200 base pairs. Finally for treatment D the first fragment migrated 19 mm which corresponds to 2322 base pairs, and the second fragment migrated with 1120. Our results for our transformation portion were as follows.
Our first tube was clear and contained TE/no DNA (negative control), second tube contained p AMP/DNA and was brown, third tube was p KAN/DNA, and the fourth tube was our unknown blue tube. All four tubes grew lawns of bacterium on the LB medium. For the clear tube, it was found that their was no growth on the mediums containing LB/AMP and the medium containing LB/KAN. For the brown tube, it was found that there were colonies found on the medium LB/AMP but none on the medium LB/KAN. For the purple tube, it was found to be no growth on the medium LB/AMP, but there was growth on the medium LB/KAN. For the unknown, there was no growth on the LB/AMP but was growth on the medium LB/KAN.
Discussion The super coiled DNA traveled the farthest in relation to its distance per base pair, even though the double digested shows to have traveled the farthest, because of its smaller fragments. The single cut, though, when looking at the photos of electrophoresis it traveled the least farthest because of its linear form and relation of distance per base pair We were able to determine the size of each fragment in the gel of our unknown by extrapolating the information from our standard curve. It was done by comparing the distance migrated to the size of the base pairs set by the lambda curve. The reason we had three separate samples was for comparison, because each of the samples compared directly to the size of the cuts given to us from the plasmid map.
Lambda sample (clear tube) was used as a positive control for it showed the true size of each fragment. The uncut sample was used as a negative control because there were no cuts, therefore no linear DNA. The S sample was our unknown, only cut once, and the D sample was also an unknown but cut twice. The biochemical test control was ethidium bromide because when it was put under UV light it fluoresces.
By being able to see each fragment we were then able to measure them and compare their sizes to the plasmid map. When comparing the plasmid map cut sizes to what was found in the electrophoresis gel, it was easy to see that the unknown sample was p KAN. When looking at plasmid map cut sizes this single cut is expecting to be 4207 base pairs, our result was 3500 base pairs, and when looking at the double cut we see that one fragment is expected to be 1875 base pairs, and the other 2332 base pairs, and it is found that the first fragment is 2322 and the second is 1120. Because of this close relation we can finalize that our unknown plasmid was p KAN. There may have been differences in the numbers due to the fact that errors may have been committed during the electrophoresis, such as, not enough buffer so the arose gel may have gotten too hot, air may have been captured within the wells when loading, contamination while during loading, and letting the gel run too long, to name a few.
We hypothesized about the transformation experiment that if there was growth on the LB/AMP medium then the unknown would be resistant to the ampicillin because it would contain p AMP plasmid, and if growth occurred on the LB/KAN then the unknown would be resistant to the kanamycin thus containing p KAN plasmid. In reference to our results, our unknown showed growth on the medium LB/KAN thus testing positive for Kanamycin, therefore our unknown plasmid WAS p KAN. Our negative control for this experiment was the clear tube containing TE/no DNA. This was used to make sure the cells would survive the competency and the preperation of transformation process, as well as ensuring that the cells were sensitive to ampicillin and Kanamycin.
There may have been some errors within this experiment. Such as, measuring errors, mislabeling, not enough shock to the E. coli, cross contamination either with hockey stick or pipette tips, and the rare occurance of spontaneous mutation. In conclusion, we found our unknown plasmid to be p KAN, because of it growth on kanamycin and its fragment sizes when compared to the plasmid cut map in its three different forms of undigested, single digested, and double digested.
Works Cited 1) Lawrence, S. M. , K. Heide mann, and D. O. Strategy. 2001.
Biological Science 111 L Laboratory Manual, 5 th edition. p. 145 - 163. Hayden-McNeil, Plymouth. 2) Rosamund, J. W, D. L. Hermann.
Biomedicine: Containment of Antibiotic Resistance. Science February 20: 98: (279): 1153 - 1154. (1998). 3) web 4) web
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