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Molecular Biology Experiment 2
Molecular Biology Experiment 2
Aim: To identify DNA sequences characteristic of the E. coli topA and cysB genes and
confirm the orientation of the topA-cysB insert within the expression vector pTrc99A/topA using
the restriction endonucleases.
Abstract:
Four reaction mixtures were made. These reaction mixtures contained some, but
not all of the following reagents-sterile water, 10 digestion buffers, plasmid DNA (125g/ml),
EcoRI (10U/l), BamHI (10U/l) and PvuII (10l). EcoRI, BamHI and PvuII are all restriction
endonucleases and were used to digest the pTrc99A/topA-cysB recombinant at various
recognition sites on the DNA molecule. After digestion of the pTrc99A/topA-cysB recombinant
it was analyzed by agarose gel electrophoresis. The results obtained indicated that the digestions
by the restriction enzymes were successful.
Introduction:
Method:
140-141.
the double digest in tube 3 using BamHI and PvuII, a lot of fragments were produced although
four fragments of similar sizes to the expected were present. The reason for these differences
may be due to the fact that the insert was cut by PvuII in tube 3 where as it was in tube 2,
therefore maybe different conformations were present for both the pTrc99A vector and the topA
insert; hence giving rise to that many number of bands. In tubes 1 and 4 only one restriction
endonuclease was used, BamHI and PvuII, respectively. Whereas BamHI was expected to cut
twice and thus produce two fragments, EcoRI was expected to cut only once and thus produce
one fragment. However, on observation of the electrophoretogram two fragments were observed.
Since the one cut that PvuII makes is that of the topA insert it means that there must be a second
PvuII site present in the pTrc99A vector. This second site was deduced to be at 7860bp.
Restriction enzymes digests aid in the sequencing of unknown DNA sequence. Because the
restriction endonucleases digestions in this experiment were successful they aided in the
identification of sequences within topA-cysB and in determining its orientation in the plasmid
sample.
References:
1. Zachary F. Burton. (1997).Experiments in Molecular Biology: Biochemical Application,
Experiment 13A pages 142-144, Restriction Endonucleases pages 58-59, Restriction Map page
48, Restriction enzyme mapping data appendix 3 pages 213-214.