( IC-R-linker/primer and LIC-L-adaptor...)Constructing Directional cDNA Libraries from Limited Amounts of RNA

The pCMV-PCR Vector

Stratagene designed the pCMV-PCR mammalian expression vector to be used with the PCR Library Construction Kit. The pCMV-PCR vector (Figure 1) is derived from the pCMV-Script vector ⁶ and features the LIC site. The pCMV-PCR vector shares many attributes of its parental pCMV-Script vector, such as the cytomegalovirus (CMV) promoter^### for constitutive expression in a wide variety of cell lines. It also includes the neomycin-phosphotransferase gene under dual control of the -lactamase and SV40 promoters, which provides selection by kanamycin resistance in bacteria and G418 resistance in mammalian cells. The multiple cloning site (MCS) of the pCMV-PCR vector is flanked by T3 and T7 promoters, which allow generation of RNA by in vitro transcription of a DNA insert in either orientation.

Fig.1

Ovarian Tumor Library

An ovarian tumor library was made from approximately 100 ng of total RNA derived from LCM tissue. cDNA was synthesized and amplified by PCR with LIC-specific primers. The PCR products were purified, prepared for LIC, annealed to the LIC-ready pCMV-PCR vector, and transformed into XL10-Gold cells. The cloning efficiency was approximately 2.0 x 10⁵ colony-forming units (cfu)/g of PCR-amplified cDNA. We examined this library by PCR analysis of 20 randomly picked colonies to determine the background levels and the distribution of insert size. The clones examined contained an average insert size of 700 bas
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