Optimised cDNA size selection and cloning procedure for the construction of representative plasmid cDNA libraries.
Plasmid libraries are more versatile than phage libraries since they allow expression cloning in eukaryotic systems. However, high numbers of primary clones are sometimes difficult to obtain and more efficient transformation procedures are often required. In this paper, a detailed protocol is presented, for the construction of large plasmid libraries from ng quantities of cDNA, based on a highly efficient transformation step. Drop dialysis and electroporation are optimised: complete ligase removal and yeast tRNA addition before dialysis appear critical, while exclusive use of double-distilled water as well as rapid preparation of fresh cells provide excellent electroporation yields. A novel, simple and flexible cDNA size selection procedure is also presented, based on sucrose density gradient. Two libraries were constructed using an expression vector for mammalian cells (pCDM8) and its Escherichia coli host strain (MC1061[p3]). Numbers of 2 to 10 x 10(6) primary transformants were obtained from 1 microgram of poly(A)+ RNA. Up to 85% of the clones had inserts and half of the inserts were larger than 1.5 kb.
Kieffer
B L
BL
Ecole Supérieure de Biotechnologie, Strasbourg, France.
D003001:Cloning, Molecular / Q000379:methods*; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005822:Genetic Vectors; D010957:Plasmids / Q000235:genetics*; D014169:Transformation, Bacterial
D004277:DNA, Single-Stranded