I. Using the DNA distribution kit to extract devices 1-3:
We filled the 4 wells we needed with 10 uL of dH2O and let it sit for about 10 minutes to resuspend. We transformed the resuspended DNA into chemically competent
E.coli cells (DH5) and plated them on an agar plate with kanamycin (plate for device 1) and chloramphenicol (plates for devices 2a, 2b/3b, and 3a) using the transformation into chemically competent E. coli cells protocol (however, we used 200 uL of competent cells per transformation rather than 100uL).
II. Liquid Cultures:
We collected our plates from the 37°C incubator the following day. All plates had colonies growing on them, so we took one from each for generating liquid cultures.
III. MiniPrep Sequencing:
Having let the cultures grow up overnight, we took samples from each culture and used the Miniprep kits to extract the plasmids. Using the Nanodrop to measure the DNA concentration we prepared them for sequencing by SourceBioscience. The 5uL sequencing samples were frozen overnight and then sent for sequencing the next day. The remaining 45uL were stored in the -20⁰c freezer.
IV. Sequencing Results:
We received sequencing results that confirmed the sequences of the parts we were using from the DNA-distribution kit, including the two point-mutations in part 3A. Shown below are the sequence alignments of the sequence we expected (top strand) and the sequencing results (bottom) of parts 2A (left) and 3A (right):
V. Restriction Digest:
The first device is already built, but devices 2 and 3 have to be built. We decided to do a restriction digest of the plasmids. We opened the plasmid with part A (promoter) using SpeI and PstI and excised the B part of its vector using XbaI and PstI, which would facilitate the ligation of XbaI-site of the B-part to the SpeI site of the promoter region. The PstI sites of both parts A and B of course would ligate together. By retaining the promoter region within the backbone we allowed for easier detection of the larger region in DNA gel electrophoresis.
VI. Ligation:
After running the gel, and clearly identifying the fragments of DNA we required, these were extracted and the appropriate combinations (2A and 2/3B; 3A and 2/3B) were ligated. These two solutions were placed under PCR and then left overnight. Measurements with the Nanodrop displayed confident concentrations of DNA (all >25ng/uL), as to be expected.
VII. Transformation and Liquid Cultures - 2:
We transformed this DNA the following day into chemically competent
E. coli cells, as in Step I. We also made 1/1000 dilutions of each plasmid (2 and 3), and spread onto plates, giving a total of four plates. The agar in this instance, in comparison to Step I, only contained chloramphenicol and not kanamycin. These revealed very positive growth after overnight incubation at 37°C, and we took the two largest colonies from each plate from which to make liquid cultures, as described in Step II. One of the colonies from the 2-dilution plate failed to continue to grow in liquid culture however.
VIII. MiniPrep and Sequencing - 2:
As before, the plasmids were extracted following the MiniPrep protocol, from each of the 7 liquid cultures. Samples were tasted in the NanoDrop (concentrations ranged between 80-170 ng/uL). 5uL samples were prepared and sent off for sequencing, to ensure that our plasmids were correct in compositions.
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