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.
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