Subcloning Of Fungal CDNA From PBK Lab

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Subcloning of fungal cDNA from pBK-CMW into a plasmid vector pUC19 using fungal gene CIH

A plasmid is a circular, double stranded DNA molecule that replicates independently of the chromosome DNA within a cell.pUC19 is one of the most commonly plasmid cloning vector used due to its high copy replication number
(approx. 100 copies per cell), ampR (ampicillin resistance gene) andterminal fragment of β -galactosidase (lac Z). It is circular double stranded and it has
2686 base pair length from which 54 are multiple cloning sites polylinker that contains unique sites for specific restriction endonucleases. pUC19 contains ampicillin resistance gene, which allows selection for bacteria that has received a copy of the
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The samples used shows the distance moved by each.
This fragment sizes were determined by comparing the unknown to a set of standard samples of known concentration, using table 2 data.

DNA fragment size of unknown base Distanced moved from well (mm) pairs

5000 15 mm 3000 20 mm 2000 27 mm 1000 32 mm 500 43 mm

Table 2: Distance moved by plasmid vectors

Distance moved Size (bp)

Band intensity

(mm) pUC19 14 mm 2659 bp


27 bp Cannot be seen pBK-­‐CMV 13 mm 4518 Bright

24 mm 600 Dull

Laboratory 2
Bacterial Transformation
Bacterial cells are plated and allowed to grow into colonies. All bacteria that will break up pUC19 will grow on ampicillin agar because pUC19 has ampicillin resistance. Transformed cell
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