Other Species Collections
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.