Generate High-Quality, Directional Plasmid cDNA Libraries ( hesis 1 is optimized using ...)

Generate High-Quality, Directional Plasmid cDNA Libraries

hesis¹ is optimized using cloned MMLV reverse transcriptase to synthesize a greater yield of high-molecular-weight cDNA. Transformation of plasmid clones is significantly improved by the use of XL10-Gold ultracompetent cells,² which generate larger primary plasmid libraries with greater complexity and more full-length clones.

Directional cDNA Synthesis

The pBluescript II, pCMV-Script and pAD-GAL4-2.1 XR library construction kits feature directional cDNA synthesis** using Stratagenes cDNA Synthesis Kit. Primary libraries containing greater than 1 x 10⁶ primary colonies are routinely achieved with 5 g of mRNA from a variety of tissues. Directional synthesis (figure 1) doubles the probability of expressing functional protein by ensuring that cDNA is in the proper orientation for protein expression. In this method, MMLV reverse transcriptase is used to synthesize first-strand cDNA from mRNA using a hybrid oligo(dT) linker-primer that contains an Xho I restriction site near its 5 end. First-strand synthesis takes place in the presence of 5-methyl dCTP and nonmethylated dATP, dGTP and dTTP. Second-strand synthesis is then performed using DNA polymerase I, cloned RNase H³ and nonmethylated dNTPs. The resulting hemimethylated cDNA is blunted using Pfu DNA polymerase,⁴ ligated to EcoR I adaptors and digested with Xho I. Since any internal Xho I sites are hemimethylated and protected from digestion, only the nonmethylated Xho I site in the linker-primer is cleaved. The resulting cDNA contains an Xho I-compatible overhang on its 3 end and an EcoR I-compatib
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( hesis 1 is optimized using ...)Generate High-Quality, Directional Plasmid cDNA Libraries

Directional cDNA Synthesis