GE 100 reactions 79770, 188403, 33, 50 reactions 79760, 50 reactions AH9539, 500 reactions, 79750 Reading farther from or closer to the primer

1min

4min

10min

20min

300 bases

200 bases

150 bases

Figure 3. Use of dITP requires longer extension times at 60°C. Shown are four sequences of plasmid pUC18 obtained using cycles with 1, 4, 10 and 20 minute extension steps in the cycles. Extension steps of 4-5 minutes or longer are necessary for reading beyond 200 bases.

Reading farther from or closer to the primer

The termination mixes described in the protocol will typically yield sequencing data from the first base to over 500 bases from the primer. This is as much sequence as most users will be able to read using current standard electrophoresis technology. If it is desired to obtain sequence >500 bases from the primer, the dNTP:ddNTP ratios can be easily altered to shift the distribution of sequencing reaction products by adding more dNTPs to the termination reaction. Adding 3∝l instead of 2∝l dNTPs will increase the dNTP:ddNTP ratio

Contents

STORAGE CONTENTS COMPONENTS OF THE KIT QUALITY CONTROL SAFETY WARNINGS AND PRECAUTIONS INTRODUCTION Chain termination sequencing Page MATERIALS NOT SUPPLIED Necessary reagents: Necessary equipment: PROTOCOL 3.Cycling termination reactions SUPPLEMENTARY INFORMATION General guidelines Preparation of template DNA Preparation of single-strandedtemplate DNA Preparation of double-strandedplasmid DNA Cycle conditions and template quantity Cycling temperatures Number of cycles and quantity of template Designing a new sequencing primer Figure Sequencing PCR Products Enzymatic pre-treatmentof PCR products Elimination of compressions Page Reading farther from or closer to the primer Denaturing gel electrophoresis Gel electrophoresis reagents Buffers Gel recipes (for 100ml of gel solution) General guidelines for electrophoresis TROUBLESHOOTING Film blank or very faint Bands faint near the primer Bands smeared Bands appear across all 4 lanes CONTROL DNA SEQUENCE REFERENCES RELATED PRODUCTS Kits and Enzymes PCR Clean-up USB Ultrapure reagents for DNA sequencing USB CORPORATION Material safety data sheet HAZARDS IDENTIFICATION FIRST-AIDMEASURES FIRE-FIGHTINGINFORMATION ACCIDENTAL RELEASE MEASURES HANDLING AND STORAGE

GE Reading farther from or closer to the primer

Models: 100 reactions 79770 188403 33 50 reactions 79760 50 reactions AH9539 500 reactions 79750

Reading farther from or closer to the primer