Coupled analysis of bacterial transformants and ligation mixture by duplex PCR enables detection of fatal instability of a nascent recombinant plasmid.
When a DNA cloning experiment fails, it is often difficult to distinguish between an inadequate cloning protocol and instability of the new recombinant plasmid. The identification of plasmid instability is particularly challenging when the instability is fatal and no DNA of the expected construct can be isolated. We have effectively addressed this problem by employment of duplex PCR (insert-insert, vector-insert) to analyse both the ligation mixture and the resultant bacterial transformants. Using this approach we found a fatal maintenance instability of one of the plasmids generated during subcloning of the cDNA for human LDLR in Escherichia coli STBL2. The described duplex PCR screening method allows monitoring of the fate of nascent recombinant plasmid from ligation, through the initial bacterial colony and the subsequent overnight culture.
Al-Allaf
Faisal A
FA
Section of Cell and Molecular Biology, Faculty of Medicine, 2nd floor, Sir Alexander Fleming Building, Imperial College London, London SW7 2AZ, UK. faisal.allaf@imperial.ac.uk
Tolmachov
Oleg
O
Themis
Michael
M
Coutelle
Charles
C
D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D053938:DNA Fragmentation; D004269:DNA, Bacterial / Q000235:genetics; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005821:Genetic Techniques; D006801:Humans; D007763:Lac Operon; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic; D013696:Temperature; D013997:Time Factors
D004269:DNA, Bacterial; D018076:DNA, Complementary; D004247:DNA