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The plasmid is 3.5 kb in size (Fig.5). It has two ECoRI restriction sites, hence should break any recombinant DNA into two fragments, i.e. ~3.5 kb and the inserted DNA.
In our experiment – There are three fragments of DNA which shows that the inserted PCR product must have one ECoRI site. The ECoRI site in the PCR product is placed almost in the middle breaking it into two fragments, viz. 600 bp and 800 bp (Fig. 6).
Interpretation - It appears that the insert has two restriction sites for NCoI (Fig 7). But position of the sites would result in a DNA fragment smaller than 1.5 kb (the total size of the insert), the size discrepancy is difficult to explain.
Interpretation – This result is totally unexpected! The bands corresponding to both the restriction enzymes, i.e. at 1.0 kb, 2.5 kb, 3.0 kb are there but in addition there are three other intermediate size DNA fragments. The bands appear to be intermediates produced due to impartial digestion of the recombinant by the two restriction enzymes.
Reason – Concentration of the enzymes was less, time of incubation is less than optimum or incubation conditions were inadequate. Sufficient time should be given for the restriction enzymes to act on all the sites.
ii. NcoI digestion (Lane 4) – the restriction sites appear to be different than mine (Fig 4a). The NcoI sites appear to be closer in the 16S rDNA extracted by this student and give rise to a DNA fragment of 600 bp.
Janda M. and Abbott S.L. (2007). 16S rRNA Gene Sequencing for Bacterial Identification in the Diagnostic Laboratory: Pluses, Perils, and Pitfalls. J. Clin Microbiol. 2007 September; 45(9): 2761–2764.
Khare N., Sharma D., Somashekar U., Prakash A., Prakash S., Mendki M.J. and Anvikar A. (2008). Detection of bacterial DNA in cholesterol gall stones. The Internet Journal of Surgery 16 (2). Available from
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It involves pasting the whole genome sequence of the organism given in a FASTA format then clicking the ORF finder that will automatically give the start and the stop codons alongside the longest pattern. In this case, the human genome was given and upon subjecting it to
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