Attempts to Construct an Enlarged pUC19 via Insertion of HindIII-digested Coliphage λ DNA

Abstract

The plasmids pUC19 and pBR322 are commonly used as vectors in molecular biology contexts. It has been observed that when Escherichia coli cells are co-transformed with these two plasmids, pUC19 is preferentially retained while pBR322 is excluded. Several hypotheses have been proposed to account for this phenomenon, one of which is the larger size of pBR322. The objective of this study was to construct an enlarged pUC19 vector (pVACC13W) via insertion of λ DNA fragments in order to test the role of size on the exclusion effect observed when pBR322 was co-transformed with pUC19 into E. coli DH5α. A HindIII restriction digest was performed on the λ genome, and the resulting fragments subjected to agarose gel electrophoresis. Two fragments, sizes 2322 bp and 2027 bp, were extracted from the gel and a ligation reaction performed at the HindIII restriction locus within the multiple cloning site of pUC19 in an attempt to produce plasmids of sizes 5008 bp and 4713 bp respectively. However, no colonies were obtained when these ligation reactions were transformed into E. coli DH5α, potentially due to a failure to inactivate T4 ligase in the ligation mixture prior to transformation or contamination of the DNA samples resulting in inefficient ligation. The results of this study indicate that isolation of λ DNA fragments via agarose gel extraction introduces the potential issues of low DNA yield and contaminated samples, suggesting that an alternate method – perhaps the amplification of desired fragments using PCR – may be more effective.