Applied Biosystems™ BigDye™ Terminator v1.1 & v3.1 5X Sequencing Buffer

Product Code.	Quantity	unitSize
15768289	1 mL	Each
15929182	1 mL	1mL
15791816	28 mL	Each
15939182	28 mL	28mL
15879776	233 mL	Each
16355292	233 mL	233mL

Product Code. 15768289 Supplier Applied Biosystems™ Supplier No. 4336697

Description

This sequencing buffer is optimized for use with BigDye™ Terminator v1.1 and v3.1 Cycle Sequencing Kits.

• Optimized for use with BigDye™ Terminator Cycle Sequencing Kits

Order Info

Each

Specifications

• Buffer: Sequencing Buffer
• Concentration: 5X
• Content And Storage: Store at 2°C to 8°C.
• For Use With (Equipment): GeneAmp 9700, Veriti Thermal Cycler, 3500 Genetic Analyzer, 3730xl DNA Analyzer, 3130xl Genetic Analyzer, 3500xL Genetic Analyzer, 3730 DNA Analyzer
• For Use With (Application): Standard Sequencing
• Product Line: BigDye
• Product Type: Sequencing Buffer
• Quantity: 1 mL
• Shipping Condition: Room Temperature
• Form: Liquid

Frequently Asked Questions (FAQs)

I am getting shorter reads than I used to. What can cause this?

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.

Can I dilute my BigDye Terminator reactions?

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.

I received BigDye Terminator v1.1 & v3.1 5X Sequencing Buffer (Cat. No. 4336697) at room temperature. Is this correct?

Yes, BigDye Terminator v1.1 & v3.1 5X Sequencing Buffer is shipped at room temperature and needs to be stored at 2-8 degrees C upon receipt.

How do I calculate the amount of BigDye Terminator v1.1 & v3.1 5X Buffer to use in the reaction?

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

Can I dilute my BigDye Terminator reaction?

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.

What is the purpose of the BigDye Terminator v1.1 & v3.1 5X Buffer in the BigDye Terminator kits?

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.

Are the BigDye Terminator 5X buffer v1.1 & v3.1 and BigDye Terminator 5X buffer v1.0 & v3.0 interchangeable?

The composition of the BigDye Terminator 5X buffer v1.1 & v3.1 and BigDye Terminator 5X buffer v1.0 & v3.0 chemistries is different and therefore should only be used with the matching chemistry. Use of the incorrect BigDye Terminator 5X buffer can negatively impact sequencing results.

For Research Use Only. Not for use in diagnostic procedures.