The experiment was to screen out which bacterial culture was successfully cloned with the recombinant DNA insert. The methods involved the use of commercial kit, QIAprep Spin Miniprep to extract and purify the plasmid DNA from the bacterial cells, followed by restriction digestion using two digestion enzymes, to cut the designated fragment that should including the DNA insert (if the cloning was success), and finally analysed the results using gel electrophoresis.
The result showed that culture A had the recombinant DNA insert; while culture B was not, as compared to the positive control included.
As in real situation, there will be no hundred percent successes in cloning, due to self-ligation that may take place in the plasmid vector. This kind of experiment is valuable and frequently employed in Molecular Science to pick out the successfully cloned bacterial colonies, and harvest them for subsequent usages. Moreover, the combination of restriction digestion and gel electrophoresis, are important techniques used in restriction mapping.
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Two bacterial cultures (A and B) were provided in this experiment, the objective was to find out which culture the plasmid DNA was containing the inserted recombinant DNA fragment (Sau3A ), and also the size of the recombinant DNA fragment.
In the first part of the experiment, a commercial kit QIAprep Spin Miniprep Kit  was used to obtain a high quality DNA for subsequent digestion. It involved the preparation and clearing of the bacterial lysate, the adsorption of DNA onto the QIAprep membrane, and the washing and elution of the plasmid DNA.
The second part involved the single or double digestion of the plasmid DNA, using the restriction enzymes XbaI and XhoI . The restriction enzymes recognized particular sequences in the plasmid DNA, pBluescript II KS , to perform the cutting. Thus, in the double digestion using both enzymes, a small fragment containing the recombinant DNA should be yielded, if the plasmid DNA is positively cloned with the recombinant DNA. This small fragment could be screen out in subsequent gel electrophoresis in part three of the experiment.
By applying an electric field to the agarose gel, the negative charge DNA molecules will migrate toward the positive charge anode. The smaller molecules, the faster or farther they will migrate. Therefore, DNA fragments of different size can be separated. [ ]
Finally, the result of the gel electrophoresis could be visualized under UV, as the agarose gel contained the Ethidium bromide (EtBr), which will intercalate with the DNA bases and fluoresce under UV, and the bands become visible.
We followed the procedure download from WebCT, and we found that there were some different in the part two and three of the experiment, comparing to the procedure distributed on the day of practical, and they were stated below.
In part one of the experiment, since the solution of culture A was quite clear, in order to increase the yield of DNA from it, we typically pelleted 1.5 ml twice in the same tube, thus obtaining a pellet from 3 ml culture.
We followed the procedures in the practical manual to lysed the bacterial cells, precipitate the genomic DNA, then applied the supernatant to the QIAprep column, to allow the plasmid DNA trap in the membrane by affinity. Then washed the column to remove trace nuclease activity and impurities, and finally we get the high quality purified DNA elutes from the column.
Then we proceeded to part two, the restriction digestion. Six tubes were setup with different digestion enzymes added for each culture as tabulated below (other ingredients and details can be referred to the practical manual):
DNA from Culture A
DNA from Culture B
XbaI and XhoI
XbaI and XhoI
N.B. The amount of 10x BSA used in each tube was 1μl instead of 2μl.
Then we proceeded to part three, the Gel Electrophoresis. The gel was setup as follows:
Well 1-10 assignment:
1kb DNA ladder
Culture A (well 2-5)
DNA cut with XbaI
DNA cut with XhoI
DNA cut with XbaI and XhoI
Always on Time
Marked to Standard
Culture B (well 6-9)
DNA cut with XbaI
DNA cut with XhoI
DNA cut with XbaI and XhoI
Control DNA with the Sau3A fragment
1 2 3 4 5 6 7 8 9 10
Direction of DNA running
Figure 1. Schematic drawing of the position and assignment of the well, and direction of DNA migration.
N.B. 2μl of loading dye instead of 4μl was added to each DNA solution before loading to the wells. And 10μl of samples and marker were loaded into the wells instead of 3μl.
When the electrophoresis was completed, the gel was put into the Gel Imaging System  to get the digital images of the gel under UV, and copied to our USB device.
Figure. 2 below, shows the original photo taken from the gel imaging system (on the right) and a diagram from the practical manual showing the 1kb DNA ladder scale for comparison (on the left).
Figure. 3 below, shows the photo after adjusting the gamma and brightness to give better readability at the target bands. Some of the bands in interested are labelled.
Recombinant DNA fragment in Culture A
Vector DNA fragment in Control
Recombinant DNA fragment in Control
By comparing to the control in lane 10, which contains the recombinant DNA fragment (the lower band), we can observe that only in lane 5 (double digestion of culture A) had a band at similar level, although it is faint, but was still quite clear. We did not observe any band at similar level in the lanes of culture B.
The result showed that culture A had the recombinant DNA insert; while culture B did not.
By comparing the 1kb DNA ladder, the target fragment in lane 5 (culture A) is about 600 base pair, by rough proportional calculation. A more accurate result can be done by plotting a graph of log size (bp) of the DNA fragment against the distance migrated (cm) 
However, in our case, the bands of the DNA ladder were not cleared and discrete enough to perform more accurate measurement.
Therefore, we can conclude that Culture A contained the target recombinant DNA fragment, Sau3A, which was about 600 base pair; while Culture B did not.
In lane 5, was the double digested DNA from culture A, which should contained a large and a small fragment. The small one was the cut fragment with the recombinant DNA insert, the Sau3A, the size should be around 700 bp, and our result was about 600 bp, closely matched with the theoretical value. While the large fragment was the remaining vector portion, the size should be about 3kb, and our result was corresponded to the position of 3054 bp.
Lane 3 and 4, were the single digested DNA from culture A, which should be linear molecules after cutting. So, the bands theoretically should be around 3700 bp, which was the sum of the size of the recombinant DNA insert and the plasmid vector. What we got in the result, was about 4000 bp, corresponded to the DNA ladder. It was also closed to the theoretical value.
Similarly, in lane 7 and 8, which were single digested DNA from culture B. Since culture B did not possess the recombinant DNA insertion, the fragments size after digestion should be about 3 kp (the size of the vector), and our result was closely matched.
From observation, the separation of bands above 3kb of the DNA marker was poor, it was difficult to have accurate measurement of the bands in that area. Also, we could observe that those bands in culture B were too bright in fluorescence making the bands too thick. This might be due to too much DNA loaded to the gel, or the electrophoresis was not long enough, that caused the bands to bunch up.
We would suggest to repeat the experiment with less amount of DNA loaded and longer electrophoresis to make the bands separate better and clearer.