BigDye™ Terminator v1.1 & v3.1 5X Sequenzierungspuffer

A change in the read length is usually caused by something that either changes the migration of the DNA or the efficiency of the sequencing reaction. Some of the things that can cause this are:

• Buffer: Buffer may be prepared incorrectly or on the system for too long.

• Polymer: Polymer may have been on the system the system too long, used past the expiration date, frozen/crystallized and thawed, or mixed with water or buffer during capillary fill. There may be a mismatch between polymer type selected in software and what is installed on the system.

• Environment: Instrument may be operating outside environmental specifications in the Site Preparation Guide. You may have poor/improper instrument ventilation, or air flow may be blowing directly on the system.

• DNA quality: RNA/protein contamination may be reducing the sequencing reaction efficiency. If working with PCR product, there may be carryover of primers and/or dNTPs from the PCR reaction, or the sequencing reaction cleanup is incomplete. If using a kit that has beads in it for the purification, beads may be blocking the capillary tip. If using BigDye XTerminator Purification, heating the XTerminator reagent can cause loss of smaller products.

• DNA quantity: There may be too much DNA competing for entry into the capillary with labeled product, or excessive amounts of DNA creating a temporary blockage of the capillary.

For more information on other causes of short reads and how to address each issue, please refer to the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C). The guide can be found by searching the Thermo Fisher Scientific website with the catalog number 4305080.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The BigDye Terminator v1.1 and v3.1 chemistries can be diluted, and a 5X Sequencing Buffer is provided with the kit to enable you to do so. However, dilution of the BigDye Terminator may compromise your data integrity depending on the cycle sequencing protocol you are following and the sequence of the particular DNA you are investigating. TheBigDye Direct Cycle Sequencing Kit and the dGTP BigDye Terminator Ready Reaction Kits v1.0 and v3.0 cannot be diluted.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The calculation can be done manually or by using our Applied Biosystems BigDye Terminator v1.1 & v3.1 5X Sequencing Buffer Calculator.

The formula is provided below:

Volume of BigDye Terminator v1.1 & v3.1 5X Buffer (µL) = 0.5 x [(total reaction volume/2.5) - volume of BigDye Terminator Ready Reaction Mix].

For example, if you are using 2 µL of the BigDye Terminator Ready Reaction Mix and a 15 µL reaction volume

Volume of BigDye Terminator v1.1 & v3.1 5X Buffer (µL) = 0.5 x [(15/2.5) - 2] = 2 µL

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

We provide the BigDye Terminator v1.1 & v3.1 5X Buffer to enable dilution of the BigDye Terminator Ready Reaction Mix. However, without optimization, diluting the BigDye Terminator Ready Reaction Mix may cause deterioration of sequencing quality. We cannot guarantee the performance of BigDye chemistry when it is diluted.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.

The BigDye Terminator v1.1 & v3.1 5X Buffer is used to maintain the optimal buffer concentration in the sequencing reaction when less than the recommended volume of BigDye Terminator Ready Reaction Mix is used. The BigDye Terminator v1.1 & v3.1 5X Buffer amount varies depending on the volume of the BigDyeTerminator Ready Reaction Mix. To determine the amount of BigDye Terminator v1.1 & v3.1 5X Buffer, please use the Applied Biosystems BigDye Terminator v1.1 & v3.1 5X Sequencing Buffer Calculator.

Find additional tips, troubleshooting help, and resources within our Capillary Electrophoresis Applications Support Center.