Linker & Primer Sequences

Linker 1 A (obtain gel-purified) 5' TTT GGA TTT GCT GGT GCA GTA CAA CTA GGC TTA ATA GGG ACA TG 3'
Linker 1 B (obtain gel-purified) 5' TCC CTA TTA AGC CTA GTT GTA CTG CAC CAG CAA ATC C[amino mod. C7] 3'
Linker 2 A (obtain gel-purified) 5' TTT CTG CTC GAA TTC AAG CTT CTA ACG ATG TAC GGG GAC ATG 3'
Linker 2 B (obtain gel-purified) 5' TCC CCG TAC ATC GTT AGA AGC TTG AAT TCG AGC AG[amino mod. C7] 3'
We now order primer 1 and primer 2 with the addition of 2 sequential biotins on the 5' end gel-purified from Integrated DNA Technologies.
Primer 1 5' GGA TTT GCT GGT GCA GTA CA 3'
Primer 2 5' CTG CTC GAA TTC AAG CTT CT 3'
Biotinylated oligo dT (obtain gel-purified)
5' [biotin]T18
M13 Forward 5' GTA AAA CGA CGG CCA GT 3'
M13 Reverse 5' GGA AAC AGC TAT GAC CAT G 3'
SAGE protocol
                
                
1. mRNA preparation
2. cDNA synthesis
3. Cleavage of biotinylated cDNA with anchoring enzyme
4. Binding biotinylated cDNA to magnetic beads
5. Ligating linkers to bound cDNA
6. Release of cDNA tags using Tagging Enzyme
7. Blunt ending released cDNA tags
8. Ligating tags to form ditags
9. PCR amplification of ditags
10. Isolation of ditags
11. Purification of ditags
12. Ligation of ditags to form concatemers
13. Cloning concatemers and sequencing