Generate High-Quality, Directional Plasmid cDNA Libraries ( le overhang on the 5 end. ...)

Generate High-Quality, Directional Plasmid cDNA Libraries

le overhang on the 5 end. After size fractionation, the cDNA is ready for directional cloning into either the pBluescript II, pCMV-Script or pAD-GAL4-2.1 vector predigested with Xho I and EcoR I (XR) restriction enzymes.

Transformation

In order to construct a complex plasmid library, synthesis of cDNA and ligation to the vector must be as efficient as possible. Ultimately, the final quality of a plasmid cDNA library is determined by transformation of the ligation products into the E. coli host. The optimal E. coli host for generating large, complex cDNA libraries was determined by comparing the primary size of cDNA libraries constructed in the pCMV-Script and pAD-GAL4 XR predigested vectors and transformed into XL2-Blue,⁵ XL10-Gold or DH10B*** cells (figure 2). The average number of primary clones obtained from transformation into XL10-Gold ultracompetent cells was 4.6-fold greater than the number of primary clones obtained from XL2-Blue cells and 22-fold greater than obtained from DH10B cells. For the tranformation control, supercoiled pUC18 was used. The transformation efficiency for XL2-Blue and XL10-Gold cells was 5 x 10⁹colony-forming unit (cfu)/g of pUC18 DNA, and transformation efficiency for DH10B cells was 1 x 10⁹cfu/g of pUC18 DNA. When comparing XL2-Blue and DH10B cells, the relative library size obtained when the pCMV-Script and pAD-GAL4 XR predigested vectors were used correlates well with the relative efficiencies observed for supercoiled pUC18 DNA. However, the relative library size obtained from transformation into XL10-Gold cells was 4.8-fold higher than predicted by the transformation effic
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( le overhang on the 5 end. ...)Generate High-Quality, Directional Plasmid cDNA Libraries

Transformation