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pSPORT-1 Library Method Overview

The laboratory of Dr. Minoru Ko constructed long-transcript enriched cDNA libraries in the pSPORT1 vector. The method involves a PCR amplification step using a 5' linker, LL-Sal4, and 3' primer, Sal4-S. Briefly, the method is as follows:

• First strand cDNA is synthesized from total RNA using Invitrogen's SuperScriptII and an oligo-dT primer with a Not I site [Invitrogen: 5'-pGACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT-3'].
• Second strand cDNA is synthesized using DNA polymerase I, and blunt ended using T4 DNA polymerase.
• Purified cDNA is ligated to linker LL-Sal4 and then amplified by long-range high fidelity PCR using Ex Taq polymerase (Takara) and primer Sal4-S.
• The cDNAs are digested with Sal I and Not I and cloned into the pSport1 vector.
• The average insert size in different libraries ranges from 2.4 to 3.6kb.

Reference:

Yulan Piao, Naomi T. Ko, Meng K. Lim, Minoru S.H. Ko: Construction of Long-Transcript Enriched cDNA from Submicrogram Amounts of Total RNAs by a Universal PCR Amplification Method. Genome Research, Vol. 11, Issue 9, 1553-1558.