Victor E. Velculescu, Lin Zhang,
Wei Zhou, Giovanni Traverso, Brad St. Croix,
Bert Vogelstein and Kenneth W. Kinzler
Johns Hopkins Oncology Center
and Howard Hughes Medical Institute,
424 North Bond Street
Baltimore, MD 21231
FAX (410) 955-0548
This protocol is provided for noncommercial use only

Table of Contents
Page
Overview 3
Methods
1. mRNA preparation 4
2. cDNA synthesis 4
3. Cleavage of biotinylated cDNA with anchoring enzyme 5
4. Binding biotinylated cDNA to magnetic beads 6
5. Ligating linkers to bound cDNA 6
6. Release of cDNA tags using Tagging Enzyme 7
7. Blunt ending released cDNA tags 7
8. Ligating tags to form ditags 8
9. PCR amplification of ditags 8
10. Isolation of ditags 10
11. Purification of ditags 12
12. Ligation of ditags to form concatemers 14
13. Cloning concatemers and sequencing 16
Appendix A: Special Reagents to order 19
Appendix B: Solutions 20
Appendix C: Gel Electrophoresis and miscellaneous methods 21
Appendix D: Linker & primer sequences 23
Appendix E: Frequently asked questions and answers 24