Optimised cDNA size selection and cloning procedure for the construction of representative plasmid cDNA libraries

Area of Science:

• Molecular Biology
• Biotechnology

Background:

• Plasmid libraries offer greater versatility than phage libraries for eukaryotic expression cloning.
• Obtaining high numbers of primary clones for plasmid libraries can be challenging, necessitating improved transformation protocols.

Purpose of the Study:

• To present a detailed protocol for constructing large plasmid libraries from nanogram quantities of cDNA.
• To optimize transformation procedures, specifically drop dialysis and electroporation, for enhanced efficiency.

Main Methods:

• Optimized drop dialysis with complete ligase removal and yeast tRNA addition.
• Optimized electroporation using double-distilled water and freshly prepared cells.
• Developed a novel sucrose density gradient-based cDNA size selection procedure.

Main Results:

• Achieved 2 to 10 x 10^6 primary transformants per microgram of poly(A)+ RNA using an expression vector for mammalian cells (pCDM8).
• Constructed two large plasmid libraries with high insert percentages (up to 85%).
• Half of the inserts were larger than 1.5 kb, indicating successful cloning of substantial cDNA fragments.

Conclusions:

• The presented protocol enables the construction of large plasmid libraries with high efficiency from minimal starting material.
• Optimized transformation and size selection methods are critical for maximizing primary clone numbers and insert size.
• This method significantly advances the ability to generate versatile plasmid libraries for eukaryotic expression cloning.