Construction and Analysis of A Directional Xenopus laevis Embryonic cDNA Library for Yeast Two-hybrid Using Modified Random Primers
2008
Here we describe a new strategy for cDNA library construction, in which
the random primers directing the first strand cDNA synthesis contain
additional d (AC) at their 5'-ends, and the linkers which will be added
to the double strand cDNA contain d(GTCG) at the 5′-ends. When
the linker is added to the 3'-end of the cDNA, a complete SalI site
d(GTCGAC) will form at the 3'-end but not at the 5′-end of the
cDNA fragment. After SalI digestion, the 3'-cohesive and the
pre-existing 5'EcoRI cohesive ends are used to introduce the double
stranded cDNA into the linearized plasmid vectors. The ligation
products were used to transform E.coli to produce a cDNA library. Using
this method, we constructed a directional Xenopus laevis embryonic
cDNA library for yeast two-hybrid. We checked the ratio of empty
vectors, the size of inserts and existence of several genes, the
results showed that cDNA library construction was successful.
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