Brad St. Croix, Victor E. Velculescu,
Lin Zhang, Wei Zhou, Giovanni Traverso,
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

Table of Contents
Page
Overview 3
Methods
1. mRNA preparation 4
2. cDNA synthesis 4
3. Cleavage of cDNA with anchoring enzyme 6
4. Ligating linkers to bound cDNA 6
5. Release of cDNA tags using Tagging Enzyme 7
6. Blunt ending released cDNA tags 8
7. Ligating tags to form ditags 9
8. PCR amplification of ditags 9
9. Isolation of ditags 11
10. Purification of ditags 13
11. Ligation of ditags to form concatemers 15
12. Cloning concatemers and sequencing 17
Appendix A: Special Reagents to order 20
Appendix B: Solutions 21
Appendix C: Gel Electrophoresis and miscellaneous methods 22
Appendix D: Linker & primer sequences 24
Appendix E: Frequently asked questions and answers 25