KEVIN
1. Plasmid Isolation
The Pqrr4+I13500 plasmid was isolated from TOP10 cells in order for them to be verified via Restriction digest. This would then be made competent in order to transform mutants in.
2. Restriction Digest
The Pqrr4+I13500 isolated plasmid was digested with XbaI and PstI enzymes, then ran on 1% gel at 100V. The following gel was obtained.
As one can see, the upper bands of the colonies 4, 5, and 6 match the upper band in the pSB2K3 size control, meaning the plasmid switch to pSB2K3 seems to have been successful. Also, the bottom bands of the colonies 4, 5, and 6 match the one in the Pqrr4+I13500 in pSB1AC3 lane, meaning that the construction seems to have worked. These two lower bands might look as if they do not match; however, if you consider the slanting of the ladders at right compared to the left, it seems that the two match.
3. Sequencing
Yesterday's sequencing of Pqrr4+B0034+K082003 had been received and analyzed. The forward sequencing did not work; however, the reverse did, and an unusual problem was noticed. K082003 had matched 100% with the sequence provided by the parts registry, and Pqrr4 matched 99%, which made sense because the Pqrr4 is almost at the 1000bp region starting from behind and polymerase often reads inaccurately after reading about 1kb, but the part in the middle, B0034, was not present. This is unusual because I have sequenced the Pqrr4+B0034 construct before constructing K082003 behind. An explanation might be that the scar did not form between Pqrr4+B0034 which caused the B0034 part to be cut out. Another reason that may explain this result may be that I simply used a non-verified isolated plasmid of Pqrr4+B0034. Either way, I would have to carry out the construction procedure from the start.
Actual matching of the sequences is soon to come, as now the sequencing website is down.
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