Guidelines for submission as per Applied Biosystems’ BigDye Terminator Cycle Sequencing protocols:
|Template||Quantity / rxn||Required |
|PCR Product:||In 1.5 ml Tubes or 8well strips or 96 well plates|
|500-1 000 bp||20-50ng||5-20ng/µL|
|1 000-2 000 bp||50-100ng||10-50ng/µL|
|> 2 000 bp||100-200ng||20-50 ng/µL|
|Single-Stranded (M13 phage)||25-50ng||25-50ng 5ng/µL|
|PLASMIDS||300ng||100-200ng/µL||In 1.5 ml Tubes or 8well strips or 96 well plates|
|BAC , Cosmid, Fosmid||0.5-1.0 µg||300ng/µL|
|*Your Specific Primers||3-5pmol||5-10 µM||5µL min||In 1.5 ml Tubes or 8well strips or 96 well plates|
The concentrations listed are necessary for speedy processing and investigators should plan on providing at least 2X the volume requirements for each sequencing reaction. Supplying extra template will ensure that sufficient material is on hand if it is necessary to re-run a reaction.
For optimum results, good quality template is essential. For sequencing, the investigator is expected to provide template samples with A260/A280 of 1.8-2.0. We can include a template quality check as a custom service prior to beginning the process. If quality is insufficient, the investigator will be notified.
Template Preparation, Quality, and Quantity Requirements
Important: All samples must be labeled with the sample name, date, and user account number. All samples must be accompanied by a sequencing request form indicating the appropriate Requested sequencing service.
OPTIMIZATION of DNA sequencing reactions and troubleshooting
If you want more information about Troubleshooting DNA sequencing reactions, you can download our complete brochure in the download center