BigDye XTerminator™ Purification Kit, 2500 Preps - FAQs

Yes, after cleaning the sequencing product using the BigDye XTerminator Purification Kit, the end product can be stored up to 10 days at either 4°C or –20°C.

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After the cycle sequencing reaction is complete, unincorporated ddNTPs can form complexes that migrate more slowly than individual ddNTPs would. These complexes can interfere with electrokinetic injection, electrophoretic separation, and data analysis. In the electropherogram these can be observed as a region containing multicolored peaks of varying sizes or blobs, depending on the efficiency of the cleanup. In BigDyeTerminator v3.1 reactions, these blobs may appear around 70–80 bp and, in severe cases, 110–120 bp. For BigDye Terminator v1.1, the blobs will either migrate ahead of the data collection time (so they will not be visible) or they may be visible at the very beginning of the run.

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

While there are many kits and methods available to clean up the extension products from a sequencing reaction, the ones that Thermo Fisher Scientific has formally tested and recommends are:

•BigDye XTerminator Purification Kit
•Ethanol precipitation
•Spin column/plate purification (e.g. Centri-Sep columns, DTR Kits from Edge Biosystems)

A list of alternative methods for purification is available in the DNA Sequencing by Capillary Electrophoresis: Applied Biosystems Chemistry Guide: Second Edition (Cat. No. 4305080, Rev. C, page 119). The guide can be found by searching the Thermo Fisher Scientific website with the Cat. No. 4305080.

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The BigDye Xterminator Solution and SAM Solution do not contain aerosols.

Find additional tips, troubleshooting help, and resources within our Nucleic Acid Purification and Analysis Support Center.

For the purification of the sequencing reaction, we recommend the BigDye Xterminator Purification Kit. For reference, the following is the link to the BigDye Xterminator Purification Kit User Guide.

Additionally, our Sanger Sequencing Guide has additional recommendations starting on page 68 for purifying the sequencing reaction.

For the PCR cleanup, prior to sequencing, we recommend ExoSAP-IT PCR Product Cleanup (Cat. No. 78250.40.UL) or ExoSAP-IT Express PCR Product Cleanup. The ExoSAP-IT express is a much quicker protocol as the incubation time is reduced.

There is one 50 mL bottle of XTerminator solution and one 250 mL bottle of SAM solution.

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

The SAM solution can precipitate and get cloudy. If this happens, we recommend gently warming the solution to 25-37 degrees C and mixing to re-dissolve the precipitate. Allow to cool to room temperature before using as warm SAM solution gives poor results. Also, if the SAM solution is mixed vigorously, please allow the foam to settle down.

We recommend preparing fresh SAM Solution and BigDye XTerminator Solution pre-mix before performing the purification and using it the same day. We do not recommend storing the pre-mix.

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

The precise ratio of liquid phase and insoluble phase in the BigDye XTerminator Solution is critical to achieving specified performance. We strongly recommend against resuspending this material and cannot guarantee the specified performance of the material if you do so. It is recommended that you use wide-bore pipette tips (>1.0 mm) when pipetting the BigDye XTerminator Solution to ensure that adequate amounts of the liquid and insoluble phase are aspirated during pipetting.

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

Yes. The precipitate should be resuspended by warming the SAM solution (remember, never heat the BigDye XTerminator Solution) to 30-37 degrees C and gently vortexing. Bubbles will form on the surface of the SAM Solution when vortexed. These will dissipate within a few minutes. Allow the SAM Solution to cool to ambient temperature before use. Do not use it while warm as using warm SAM Solution will produce poor results.

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

The BigDye XTerminator Solution is expected to perform to specification if left out for no more than one 24-hour period. We cannot guarantee performance for reagent left out for a longer period of time.

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

The BigDye XTerminator Solution is expected to perform to specification if frozen no more than once.

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

The BigDye XTerminator run modules can be downloaded using the following link (http://www.thermofisher.com/us/en/home/technical-resources/software-downloads/bigdye-xterminator-purification-kit.html).

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

For a 384-well plate, a minimum 5 µL reaction volume should be used. For a 96-well plate, a minimum 10 µL reaction volume should be used.

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No. BigDye XTerminator Solution is heat-sensitive and should never be heated.

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The recommended vortexers for the BigDye XTerminator Purification Kit are the following:

- Digital Vortex-Genie 2
- IKA MS3 Digital
- IKA Vortex 3
- Taitec MicroMixer E-36
- Union Scientific Vertical Shaker


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

Because of the inconsistency that may occur with manually cutting pipet tips, we do not recommend it. However, if it is needed in the short term, while waiting for your order of wide-bore pipet tips, the tip orifice should be >1.0 mm.

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

The wide-bore tips are necessary when using the BigDye XTerminator Purification Kit to ensure that the correct amount of BigDye XTerminator Solution is used in the purification. The wide-bore tips are not necessary for pipetting the SAM Solution. If wide-bore tips are not used, an inadequate amount of BigDye Xterminator Solution may result and lead to uneven distribution of the BigDye Xterminator Solution from well to well. This may result in the liquid level of the BigDye Xterminator Solution to be consumed faster and result in a viscous solution that is difficult to pipet.

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

We recommend that you use wide-bore pipet tips (>1.0 mm) for pipetting the BigDye XTerminator Solution.

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

There are many advantages to using the BigDye XTerminator Purification Kit including:

-Simplifies workflow:
1.No need for spin column or ethanol precipitation
2. Fast
3. Little hands-on time needed
-Flexible throughput with purification in single tubes, 96-well or 384-well formats
-No bead removal prior to injection (depending upon the instrument used for sequencing)
-Efficient removal of “blobs”
-No danger of removing sample pellet (as can happen with ethanol precipitation)
-Higher signal intensity for the purified sample decreases quantity of DNA required

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

The BigDye XTerminator Purification Kit sequesters cycle-sequencing reaction components such as salt ions, unincorporated dye terminators, and dNTPs, to prevent their co-injection with dye-labeled extension products into a capillary electrophoresis DNA analyzer. The SAM Solution also stabilizes the sample after purification.

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

The BigDye XTerminator Purification Kit is intended to purify the DNA sequencing reaction after thermal cycling.

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

Sequencing reaction purification can be performed with BigDye XTerminator, Centri-Sep System or ethanol/EDTA precipitation. The protocols can be found in the BigDye Xterminator Purification Kit manual (https://tools.thermofisher.com/content/sfs/manuals/cms_042772.pdf), Chapter 4 of the BigDye Terminator v1.1 Cycle Sequencing Kit manual (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN1000356-BDTv1-1CycleSeqKit-UG.pdf), BigDye Terminator v3.1 Cycle Sequencing Kit manual (http://tools.thermofisher.com/content/sfs/manuals/cms_081527.pdf) or the DNA Sequencing by Capillary Electrophoresis Guide (http://tools.thermofisher.com/content/sfs/manuals/cms_041003.pdf).

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

For sequencing reactions, it is only necessary to remove the fluorescently labeled ddNTPs as they can interfere with the fluorescent sequencing detection. For sequencing, the extension products are single stranded and their lengths will vary, starting with primer +1, +2, +3 etc. Depending on the method, PCR cleanup methods are intended to remove primers or products below a certain basepair length, and loss of the smaller sequencing extension products can occur if a PCR purification method is used to purify the sequencing reaction.

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

Purification of the sequencing reaction gets rid of salt ions, unincorporated dye terminators, and dNTPs. These components can compete with the sample for injection into the capillary and obscure data in the early part of the sequence and can interfere with basecalling.

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