BioSample

Using 5 ug of polyadenylated mRNA from 3 day-old Arabidopsis thaliana (Columbia) seedling hypocotyls as template and oligo d(t) as primer, first strand synthesis was catalyzed by Moloney murine leukemia virus reverse transcriptase (Pharmacia). Second-strand cDNA was made using the procedure of Gubler and Hoffman (1983) except that DNA ligase was omitted. After the second strand reaction, the ends of the cDNA were made blunt with Klenow fragment and EcoRI/NotI adapters (Pharmacia) were ligated to each end. The cDNA was purified from unligated adapters by spun-column chromatography using sephacryl s-300 and size-fractionated on a 1% low melting point mini-gel. Size selected cDNAs (2 - 3 kb) were removed from the gel using agarase (New England Biolabs), phenol:choloroform extracted and precipitated using 0.3 M NaOAc (pH 7)/ethanol. A portion of each cDNA size-fraction (0.1 ug) was co-precipitated with 1 ug of lambdaZapII (StratageneEcoRI digested, dephosphorylated arms and then ligated in a volume of 4 ul overnight. Each ligation mix was packaged in vitro using Gigapack II gold packaging extract (Stratagene). We have determined that although first strand cDNA synthesis was initiated using dT, almost all of the cDNAs begin 8-10 bp from the poly-A tail. The reason for the loss of the poly-A tail is most likely due to lower than anticipated nucleotide levels during the Klenow repair of ragged ends before the addition of linkers (3'-5' exo instead of 5'-3' pol). When this library is used please reference the ABRC and: Kieber, J. et al. (1993) Cell 72:427-441.