Frequently Asked Questions - Dye Terminator Removal

You are here

What Clients are Saying:

Very Happy with Edge customer service staff.

No, the gel matrix is dispensed in deionized water.

Yes. A few hours on the bench or overnight storage will not damage performance or reduce shelf life as long as the plates are stored in their original packaging. We do not recommend long-term storage at room temperature.

Frozen plates cannot be used. The matrix ruptures and seals the base of the unit resulting in total sample loss.

Yes. For best results use no more than 0.5X BigDye® v 3.1 concentrations, e.g. 3 µL of BigDye® v 3.1 per 15µL reaction.

The radius is equal to the distance in millimeters between the axis of rotation and the bottom of the gel bed when the plate and the receiver are placed in the plate carrier in the centrifuge bucket.

To achieve RCF = 850 x g: rpm = 27,549/√r

Unincorporated dye terminators co-migrate with sequencing products during electrophoresis. The most common peaks occur roughly 70 bases into the sequencing run, and in cases of severe contamination additional peaks may appear in the vicinity of base 120, 220, or higher. Unincorporated dye terminators can appear in purified products for a variety of reasons.

(a) Unincorporated dye terminators will co-migrate with the sequencing products if the sample is allowed to pass down the side of the well rather than through the gel matrix; always use care when applying the sample drop-wise to the center of the gel matrix.

(b) The plates may have been frozen. Freezing irreversibly damages the integrity of the gel matrix and samples processed on these plates will fail.

(c) Centrifugation conditions are essential to the optimum performance of this product. Make sure that the rpm has been calculated correctly and that the spin times are correctly entered. If conditions are correct, it may be necessary to optimize centrifugation protocol for your specific centrifuge, following recommendations described in answers to 4b and 5b.

The recommended protocol assumes that timing starts when the centrifuge starts spinning. Acceleration and deceleration times are highly variable and on some centrifuges they are controllable and on others, they are not. The recommended protocol is an approximation of the best conditions for using this product. It may be necessary to adjust the spin conditions slightly from the recommended protocol to achieve optimum results.

Pre-spin the plate at 850 x g for 3 minutes, load the samples onto the plate and spin at 850 x g for 5 minutes.

Spin the plate for an additional minute at the correct g force. Even though this procedure may "save" your sample, it will not be optimal. It will likely have low signal strength and may be contaminated with unincorporated dye terminators. You may need to repeat the sequencing reaction.

(a) The plate may be dehydrated. To avoid this, plates should be stored at 4° C in their original packaging.

(b) Centrifugation conditions are essential to the optimum performance of this product. If the centrifuge is new or different, or you are a first-time user of the product, be aware that not all centrifuges respond the same to any given set of instructions. Acceleration and deceleration rates are often different and this may affect your results. First, check the spin conditions and make sure that the rpm has been calculated correctly and the spin times are correct. If the calculations are correct, then consider that centrifuges with fast acceleration rates are particularly susceptible to this type of problem. Try decreasing the length of the initial spin by 30-60 seconds.

(a) Your plate may have been frozen. Freezing and thawing usually results in higher than normal recovery volumes. Freezing will also negatively affect the performance of this product.

(b) Centrifugation conditions are essential to the optimum performance of this product. If the centrifuge is new or different, or you are a first-time user of the product, be aware that not all centrifuges respond the same to any given set of instructions. Acceleration and deceleration rates are often different and this may affect your results. First, check the spin conditions and make sure that the rpm has been calculated correctly and the spin times are correct. If the calculations are correct, then consider that centrifuges with slow acceleration rates are particularly susceptible to this type of problem. Try extending the length of the initial and final spin by 1 minute.

The volume of the purified product should be approximately 1-4 µL higher than that of the initial sample.

For best results, we recommend using volumes of 10 – 15 µL. Smaller volumes loaded onto the column will result in low signal strength, loss of 5' sequence, and shorter read lengths. Reactions < 10 µL should be adjusted to 10 µL before loading on the plate.

The recommended maximum volume is 15 µl. Larger volumes loaded onto the plate may result in dye terminator and salt contamination. This product is not recommended for cleaning BigDye® v3.1 concentrations greater than 0.5X (e.g. 3µL BigDye® v 3.1 per 15 µl reaction). Use the Performa® DTR 96-Well Standard Plate for 20 µL reactions with high dye terminator concentrations.

No, the gel matrix is dispensed in deionized water.

Yes. A few hours on the bench or overnight storage will not damage performance or reduce shelf life as long as the plates are stored in their original packaging. We do not recommend long-term storage at room temperature.

Frozen plates cannot be used. The matrix ruptures and seals the base of the unit resulting in total sample loss.

Yes. For best results use no more than 0.5X BigDye® v 3.1 concentrations, e.g. 4 µL of BigDye® v 3.1 per 20µL reaction.

The radius is equal to the distance in millimeters between the axis of rotation and the bottom of the gel bed when the plate and the receiver are placed in the plate carrier in the centrifuge bucket.

To achieve RCF = 850 x g: rpm = 27,549/√r

Unincorporated dye terminators co-migrate with sequencing products during electrophoresis. The most common peaks occur roughly 70 bases into the sequencing run, and in cases of severe contamination additional peaks may appear in the vicinity of base 120, 220, or higher. Unincorporated dye terminators can appear in purified products for a variety of reasons.

(a) Unincorporated dye terminators will not be efficiently removed if the sample is allowed to pass down the side of the well rather than through the gel matrix; always use care when applying the sample drop-wise to the center of the gel matrix.

(b) The plates may have been frozen. Freezing irreversibly damages the integrity of the gel matrix and samples processed on these plates will fail.

(c) Centrifugation conditions are essential to the optimum performance of this product. Make sure that the rpm has been calculated correctly and that the spin times are correctly entered. If conditions are correct, it may be necessary to optimize centrifugation protocol for your specific centrifuge, following recommendations described in answers to 4b and 5b of Sample Recovery section.

The recommended protocol assumes that timing starts when the centrifuge starts spinning. Acceleration and deceleration times are highly variable. The recommended protocol is an approximation of the best conditions for using this product. It may be necessary to adjust the spin conditions slightly from the recommended protocol to achieve optimum results.

Pre-spin the plate at 850 x g for 5 minutes, load the samples onto the plate and spin at 850 x g for 5 minutes.

Spin the plate for an additional minute at the correct g force. Even though this procedure may "save" your sample, it will not be optimal. It will have low signal strength and may be contaminated with unincorporated dye terminators. You may need to repeat the sequencing reaction.

(a) Your plate may have been frozen. Freezing and thawing usually results in higher than normal recovery volumes. Freezing will also negatively affect the performance of this product.

(b) Centrifugation conditions are essential to the optimum performance of this product. If the centrifuge is new, or you are a first-time user of the product, be aware that not all centrifuges behave the same to any given set of instructions. Acceleration and deceleration rates are often different and this may affect your results. First, check the spin conditions and make sure that the rpm has been calculated correctly and the spin times are correct. If the calculations are correct, then consider that centrifuges with slow acceleration rates are particularly susceptible to this type of problem. Try extending the length of the initial and final spin by 30 seconds.

The final volume of the purified sample should be approximately 1-4 µl higher than that of the initial sample.

The recommended minimum volume is 5 µl. Smaller volumes loaded onto the plate may result in low signal strength, loss of 5’ sequence, and shorter read lengths.

The recommended maximum volume is 20 µl. Larger volumes loaded onto the plate may result in dye terminator and salt contamination.

No, the gel matrix is dispensed in deionized water.

Yes. A few hours on the bench or overnight storage will not damage performance or reduce shelf life as long as the plates are stored in their original packaging. We do not recommend long-term storage at room temperature.

Frozen plates cannot be used. The matrix ruptures and seals the base of the unit resulting in total sample loss.

Yes. For best results use no more than 0.5X BigDye® v 3.1 concentrations, e.g. 2 μL of igDye® v 3.1 per 10μL reaction.

the gel bed when the plate and the receiver are placed in the plate carrier in the centrifuge bucket.

To achieve RCF = 850 x g: rpm = 27,549/√r

Unincorporated dye terminators co-migrate with sequencing products during electrophoresis. The most common peaks occur roughly 70 bases into the sequencing run, and in cases of severe contamination additional peaks may appear in the vicinity of base 120, 220, or higher. Unincorporated dye terminators can appear in purified products for a variety of reasons.

(a) Unincorporated dye terminators will co-migrate with the sequencing products if the sample is allowed to pass down the side of the well rather than through the gel matrix; always use care when applying the sample drop-wise to the center of the gel matrix.

(b) The plates may have been frozen. Freezing irreversibly damages the integrity of the gel matrix and samples processed on these plates will fail. Centrifugation conditions are essential to the optimum performance of this product.

(c) Make sure that the rpm has been calculated correctly and that the spin times are correctly entered. If conditions are correct, it may be necessary to optimize centrifugation protocol for your specific centrifuge, following recommendations described in answers to 4b and 5b of the Sample Recovery section.

The recommended protocol assumes that timing starts when the centrifuge starts spinning. Acceleration and deceleration times are highly variable and on some centrifuges they are controllable and on others, they are not. The recommended protocol is an approximation of the best conditions for using this product. It may be necessary to adjust the spin conditions slightly from the recommended protocol to achieve optimum results.

(a) For 5 μl Reaction Volumes: Pre-spin the plate at 850 x g for 2 minutes, load the samples onto the plate and spin at 850 x g for 5 minutes.

(b) For 10 μl Reaction Volumes: Pre-spin the plate at 850 x g for 2 minutes, load the samples onto the plate and spin at 850 x g for 2 minutes.

Spin the plate for an additional minute at the correct g force. Even though this procedure may “save” your sample, it will not be optimal. It will likely have low signal strength and may be contaminated with unincorporated dye terminators. You may need to repeat the sequencing reaction.

(a) The plate may be dehydrated. To avoid this, plates should be stored at 4° C in their original packaging.

(b) Centrifugation conditions are essential to the optimum performance of this product. If the centrifuge is new or different, or you are a first-time user of the product, be aware that not all centrifuges behave the same to any given set of instructions. Acceleration and deceleration rates are often different and this may affect your results. First, check the spin conditions and make sure that the rpm has been calculated correctly and the spin times are correct. If the calculations are correct, then consider that centrifuges with fast acceleration rates are particularly susceptible to this type of problem. Try decreasing the length of the initial spin by 30-60 seconds.

(a) Your plate may have been frozen. Freezing and thawing usually results in higher than normal recovery volumes. Freezing will also negatively affect the performance of this product.

(b) Centrifugation conditions are essential to the optimum performance of this product. If the centrifuge is new or different, or you are a first-time user of the product, be aware that not all centrifuges behave the same to any given set of instructions. Acceleration and deceleration rates are often different and this may affect your results. First, check the spin conditions and make sure that the rpm has been calculated correctly and the spin times are correct. If the calculations are correct, then consider that centrifuges with slow acceleration rates are particularly susceptible to this type of problem. Try extending the length of the initial and final spin by 30 seconds minute.

The final volume of the purified sample should be approximately 1-4 μL higher than that of the initial sample.

The recommended minimum volume is 5 μL. Smaller volumes loaded onto the column will result in low signal strength, loss of 5’ sequence, and shorter read lengths.

We recommend the Edge BioSystems’ MagWell™ Magnetic Separator 96, Catalog #57624. The unique configuration of the magnet (milled cavities aligned on either side of an imbedded bar magnet) places the tip of the wells below the top of the bar magnets on the plate. This configuration maximizes the magnetic field strength experienced by each well while allowing the magnetic particles to be pulled both to the side and away from the bottom of the plate. In so doing, it permits effective washing of the particles in both magnetic and manual liquid handling protocols. It also permits the user to resuspend in a minimum volume of fluid e.g. 10 μl. If using an alternative magnet, the protocol will need to be optimized by the user.

The product may be stored at 4°C, but be sure to bring the resin to room temperature prior to use. Avoid freezing the product.

Remove the sample from the resin, and store at - 20°C.

Yes, but only after completing the wash steps. The samples should be sealed to prevent overdrying and stored at 4°C. This has been tested for up to one week storage. At this time we do not recommend storage for longer periods.

This is not generally recommended since the presence of particulates in the sample carries the risk of clogging the capillaries in the DNA analyzers.

No. However, we recommend that users of this sample purification system follow the instrument manufacturer’s recommendation for the appropriate solvent to be used in sample injection. If the sample must be in formamide, the sample can be eluted in water then dried down and resuspended in formamide.

Resin is optimally dry when there is no visual evidence of residual ethanol following the drying step.

Yes. Over dried resin will be attached to the plate well wall in an obvious manner and will be difficult to work with during resuspension. Over drying of the resin may result in reduced signal strength and an increase in the overall failure rate. To avoid over drying, aspirate all ethanol, and reduce drying time at room temperature to 3 minutes, or heat at 98°C for 30 seconds. Resuspend immediately. If samples appear over dried at the elution step, add the recommended volume of deionized water, and incubate at room temperature for one minute. Pipet mix to resuspend.

Add an extra aspiration step after each ethanol removal, aspirating as slowly as possible with the pipet offset to150 μl. If this doesn’t solve the problem, then try adding a third wash step.

Blobs or unincorporated dye terminator peaks are usually a result of incomplete removal of ethanol that may contain unincorporated dye terminators. If residual ethanol is present following the first aspiration, a second aspiration is recommended. Offset the pipet to 150 μl, and aspirate as slowly as possible to remove all residual ethanol. If this step alone does not solve the problem, then try adding a third wash step to the process.

Yes. In most cases, residual ethanol from the wash steps will contain trace amounts of unincorporated dye terminators. Residual ethanol must be removed to obtain optimal results.

The optimum concentration of ethanol is 80%. Concentrations as low as 60% and as high as 90% have been tested. These other concentrations are reasonably effective at washing the resin. Lower concentrations of ethanol tend to reduce signal strength and read length especially for samples containing small amounts of DNA. Increased ethanol concentrations are less effective at washing away salts and unincorporated dye terminators leading to an increased frequency of dye blob contamination, noise, and reduced signal strength.

While one wash may be sufficient for very low dye volumes, two washes give far more reproducible results over a range of dye volumes up to 4 μl. A third wash step can increase the consistency of results when using higher dye volumes.

Yes, including Beckman DTCS™ and Amersham dyes.

The optimum amount of resin to use is 4 ul, and it has been our observation that this volume is the optimum over a broad range of conditions.

No. Premixing the MagDTR resin with ethanol negatively impacts the performance of the product.

Yes. However, 5 volumes of 80% ethanol are required to bind the sequencing reaction products. This large increase in total solution volume may obviate the advantages of using a single alcohol concentration for all steps.

Vortex mixing during the protocol process is not recommended.

No. A minimum of 15 pipet mixes gives consistent purity and yields.

If using a multichannel pipet reservoir, resin can be resuspended by rocking the reservoir back and forth 7-10 times.

Shake well or vortex the resin container until the resin is in complete suspension. For all subsequent pipetting steps, use a pipet to mix the suspension prior to dispensing into the sample.