Double-stranded PCR products, purified using an ExoSAP-IT (USB), were subsequently used for direct cycle sequencing with dye-labeled terminators (Applied Biosystems).
All short PCR products were purified using an ExoSAP-IT purification kit (USB Corporation, Cleveland, OH, USA) and then sequenced with dye-labelled terminators (Applied Biosystems) using the same primers as for PCR.
Double-stranded PCR products, purified using a ExoSAP-IT (USB), were subsequently used for direct cycle sequencing with dye-labeled terminators (Applied Biosystems).
The amplified DNA fragments were purified using a ExoSAP-IT® reagent (USB Corporation, Cleveland, OH, USA) diluted 1 20 according to the manufacturer's instructions.
Prior to cloning or sequencing, 20 μl of PCR product was purified using the ExoSAP protocol with 0.72 μl shrimp alkaline phosphatase (SAP) and 1.44 μl exonuclease I (ExoI) (Amersham Pharmacia, Piscataway, NJ).
Prior to cloning, 15 μl of PCR product was purified using the ExoSAP protocol with 0.72 μl shrimp alkaline phosphatase (SAP) and 1.44 μl exonuclease I (ExoI) (Amersham Pharmacia, Piscataway, NJ). Isolation of SINE flanked fragments was completed using the TOPO TA Cloning kit (Invitrogen).
A 1029 bp PCR product specific to NANOG, amplified with primer pair F3/R1 (Table 1), and a 990 bp PCR product specific to NANOGP8, amplified with primer pair F4/R1, were purified using a standard ExoSAP (exonuclease I/shrimp alkaline phosphatase) protocol and single-pass sequenced directly from PCR products using the F3 primer for NANOG and the F4 primer for NANOGP8.
Biopsies were taken using a standardized protocol.
For both introns, unincorporated primers and dNTPs were removed enzymatically using the ExoSAP-IT protocol.
