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pExpress-1 Library Method

Non-normalized and normalized libraries in pExpress-1 were constructed at Open Biosystems (Huntsville, AL). Briefly, the methods were as follows:

  • mRNA was isolated from total RNA using magnetic oligo-dT beads which contain a Not I site.
  • First strand cDNA was synthesized using commercially available MMLV RT (H+).
  • Second strand cDNA was synthesized.
  • The double stranded cDNA was digested with Not I and Eco RV, size fractionated (>1 KB), and cloned directionally into the pExpress-1 vector.
  • The vector was transformed into T1 bacteriophage resistant E. coli.
  • This produced the non-normalized library.
  • The initial mRNA sample was biotinylated and then used to prepare the normalized library by hybridizing the bio-RNA to the ssDNA to a Cot value of 5.