Other Species Collections
pCMV-SPORT6.1 Library Method
Non-normalized and normalized libraries in pCMV-SPORT6.1 were constructed at Invitrogen Corp., Carlsbad, CA, which also prepared the vector.
the methods were as follows:
Non-normalized (standard) libraries
- First strand cDNA was synthesized from polyA+mRNA using Invitrogen Superscript II RT and an oligo-dT primer
containing a Not I site.
- Second strand cDNA was synthesized.
- The cDNA was "polished" with T4 polymerase, digested with Not I to create 5'-blunt/3'-Not I cDNA,
then size-fractionated on a gel, purified, and ligated into the pCMV-Sport6.1 vector.
- The standard library was prepared.
- The library was hybridized using Cot-7 to Cot-25 values to reduce abundant sequences and
increase the possibility of isolating rare or novel transcripts from the library.
- Because normalization was performed on pre-constructed libraries that had been rendered
single-stranded, the average insert size and library complexity was maintained.
control of the normalized library was performed by comparing hybridization between standard
and normalized library to confirm an average reduction of at least 10 fold for the abundant