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Products - Purification Kits - Dye Terminator Kits for Single Column and Plate-Based Sequencing Reaction Purifications

dye-terminator
ArrayIt® Dye Terminator Clean-Up Kits use advanced separation chemistry and membrane binding for rapid and affordable purification of dye terminator sequencing products. The single column, 96-well and 384-well kits provide sequence data that is >99.9% accurate, and templates purified with our kits vastly outperform competing kits and alcohol precipitation methods. In the era of large-scale DNA sequencing projects and genotyping by microarray, ArrayIt® DTC kits should be used to improve sequencing and genotyping data quality.

Table of Contents

  • Introduction
  • Quality Control
  • Product Description
  • Technical Assistance
  • Short Protocols  (vacuum and centrifugation)
  • Complete Protocols (vacuum and centrifugation)
  • Short Protocols (centrifugation)
  • Complete Protocols (centrifugation)
  • Equipment Requirements
  • Troubleshooting Tips
  • Kit Contents
  • Ordering Information
  • Storage Conditions
  • Warranty

Introduction
Congratulations on taking a big step towards improving the economies of scale, quality and speed of your genomics research. This booklet contains a complete set of protocols outlining the steps and principles needed to use the ArrayIt® brand Dye Terminator Clean-Up Kits.

Quality Control
Arrayit assures the performance of this product.  The finest scientific research went into the development of this product.  Rigorous quality control monitoring on a lot-by-lot basis guarantees that the plates, buffers, and protocols conform to the highest industry standards.

Product Description
The ArrayIt® brand Dye Terminator Clean-Up Kits use sophisticated separation chemistry and 96- or 384-well purification plates to yield the cleanest sequencing products possible.  Our Dye Terminator Clean-Up Kits increase the quality of sequencing data by removing unwanted salts, enzymes, primers, unincorporated dyes and nucleotides, and other contaminants that diminish the quality of DNA sequence reads and elevate miscalls.

Users will appreciate the following features:

  • Supports all gel and capillary electrophoresis sequencing platforms
  • Industrial sequencing capacity (up to 250,000 nt per 384-well plate)
  • Reduces cost ($0.15 per run for the 384-well format)
  • Longer reads producing 10-20% more sequence data per run
  • High-throughput of 10,000 samples/day manually
  • Compatible with all 96- and 384-well thermal cyclers
  • Superior chemistry provides >99+% purity
  • Dye removal produces clean sequence data
  • Minimizes wear and clogging of costly glass capillaries
  • High yield produces stronger sequencing signals
  • No alcohol precipitations required
  • Arrives ready to use, no buffer or column preparation required

Technical Assistance
Please contact us if you have any comments, suggestions, or if you need technical assistance.  By electronic mail: arrayit@arrayit.com (under the subject heading please type ArrayIt technical assistance).  By telephone: (408) 744-1331, Monday—Friday PST 9:00am - 4:30pm. Please remember that we want to hear about your successes!

dye-terminator-cleanup
Figure 1. Sequence data from a single dye terminator sequencing reaction purified by ethanol precipitation (left) or with a 384-well Dye Terminator Clean-Up Kit (right). Superior sequence data quality early in the read is easily observed in the right panel.

dye-terminator-kit
Figure 2.  Sequence data generated with the ABI 3700 sequencer (Applied BioSystems), with templates purified with the ArrayIt® brand Dye Terminator Clean-up Kit. The extended sequence reads beyond 600 nt are readily observed.

Short Protocol (vacuum and centrifugation)

  1. Set up 5-25 Ál dye terminator sequencing reactions.
  2. Amplify sequencing templates by thermal cycling.
  3. Add 2 volumes (10-50 Ál) of Binding Buffer per sample.
  4. Mix extension products and Binding Buffer by pipetting up and down 10 times.
  5. Transfer samples into 96- or 384-well Clean-Up Filters.
  6. Bind samples to membrane using a gentle vacuum.
  7. Wash bound samples 3X with 80 Ál per well of Wash Buffer.
  8. Apply strong vacuum for 5 min to dry Clean-Up Filter membrane.
  9. Remove residual Wash Buffer by centrifugation into the Wash Microplate (5 min at 500 x g)
  10. Add 50 Ál dH20 to each well of the Clean-Up Filter.
  11. Incubate Clean-Up Filter for 1 min at room temperature.
  12. Elute purified products into a 96- or 384-well Sample Microplate (5 min at 500 x g).
  13. Evaporate samples to dryness by vacuum centrifugation (20 min).
  14. Re-suspend each sample in 3.0 Ál loading buffer and load onto sequencing gel.

Complete Protocol (vacuum and centrifugation)

  1. Set up 5-25 Ál dye terminator sequencing reactions.  Use standard equipment, kits and protocols to carry out the primer extension reactions. Reaction volumes can be between 5 and 25 Ál. The DTC kits support all standard chemistries.
  2. Amplify sequencing templates by thermal cycling. Use standard equipment, kits and protocols for this step. The DTC kits support all standard thermal cyclers.
  3. Add 2 volumes (10-50 Ál) of 2X Binding Buffer per sample.  This step can be performed manually or with an automatic liquid handling device.  Make sure not to splash reactions from well to well.
  4. Mix extension products and Binding Buffer by pipetting up and down 10 times. This step ensures efficient precipitation and attachment of the extension products to the Clean-Up Filter membrane.  Failure to mix extension products and Binding Buffer will reduce yield and produce weaker sequence reads.
  5. Transfer samples into 96- or 384-well Clean-Up Filters.  This can be performed manually by pipette, or with an automated liquid handling robot. Make sure to transfer samples accurately and without splashing.
  6. Bind samples to membrane using a gentle vacuum.  Use a vacuum manifold to apply a gentle vacuum to the Clean-Up Filter.  The salts, enzymes, nucleotides, primers and other contaminants will pass through the membrane, leaving the extension products attached to the membrane.
  7. Wash bound samples 3X with 80 Ál per well of Wash Buffer.  This step removes residual contaminants from the Clean-Up Filter membrane. Use a gentle vacuum for the three wash steps.
  8. Apply strong vacuum for 5 min to dry Clean-Up Filter membrane. This step removes most of the Wash Buffer from the membrane, leaving the extension products attached.  Do not dry for more than 5 min, as this may cause permanent binding of the extension products and reduced yield.
  9. Remove residual Wash Buffer by centrifugation into the Wash Microplate (5 min at 500 x g).  This step removes any remaining Wash Buffer, leaving the extension product on the membrane. Do not spin longer than 5 min, as this may reduce yield.
  10. Add 50 Ál dH20 to each well of the Clean-Up Filter. Use a manual or automatic liquid handling robot for this step. The dH20 solubilizes the extension product.
  11. Incubate Clean-Up Filter for 1 min at room temperature.  This step allows time for the re-suspension of the extension products. Shorter incubation times may reduce yield.
  12. Elute purified products into a 96- or 384-well Sample Microplate (5 min at 500 x g). Place the Sample Microplates beneath the Clean-Up Filter plate, and elute the samples by centrifugation.
  13. Evaporate samples to dryness by vacuum centrifugation (20 min).  This step removes the dH20 from the extension products, leaving the purified extension products as a dried pellet at the bottom of the well of the Sample Microplate.
  14. Re-suspend each sample in 3.0 Ál loading buffer and load onto sequencing gel.  Make sure to re-suspend sample completely before loading.

Short Protocol (centrifugation)

  1. Set up 5-25 Ál dye terminator sequencing reactions.
  2. Amplify sequencing templates by thermal cycling.
  3. Add 2 volumes (10-50 Ál) of Binding Buffer per well.
  4. Mix extension products and Binding Buffer by pipetting up and down 10 times.
  5. Pipette each sample into an individual well of a 96- or 384-well Clean-Up Filter.
  6. Centrifuge for 1 min (500 x g) to remove unbound material.
  7. Add 80 Ál per well of Wash Buffer and centrifuge for 1 min (500 x g).
  8. Wash the Clean-Up Filter two more times with 80 Ál Wash Buffer.
  9. Centrifuge for 5 min (500 x g) to remove residual Wash Buffer.
  10. Add 50 Ál dH20 to each well of the Clean-Up Filter.
  11. Incubate Clean-Up Filter for 1 min at room temperature.
  12. Elute purified products into a 96- or 384-well Sample Microplate by centrifugation for 5 min (500 x g).
  13. Evaporate samples to dryness by vacuum centrifugation (20-40 min). This step removes the dH20 from the extension products, leaving the purified extension products as a dried pellet at the bottom of the well of the Sample Microplate. Use elevated temperatures (e.g. 65░C) to speed up the drying step.
  14. Re-suspend each sample in 3.0 Ál loading buffer and load onto sequencing gel.  Make sure to re-suspend sample completely before loading.  Load the entire 3.0 Ál sample onto a gel and carry out electrophoresis according to the instructions of the manufacturer.

Complete Protocol (centrifugation)

  1. Set up 5-25 Ál dye terminator sequencing reactions.  The standard sequencing reaction using the ABI Prism Big Dye Terminator Kit is 20 Ál, which is made by mixing 8.0 Ál of Teminator Ready Reaction Mix and 12.0 Ál of template and primer. To conserve reagents, smaller volume reactions (5-10 Ál) can be used. Typical amounts of template are 50-200 ng for single-stranded DNA, 200-500 ng for double-stranded DNA, and 50-100 ng of a 1 kb PCR product. Typical amounts of primer are 2-5 pmoles. Custom primers may require some optimization.
  2. Amplify sequencing templates by thermal cycling. Use a 96- or 384-well thermal cycler.  Set the thermal cycler to the appropriate volume (5-25 Ál) and cycle for 25 rounds. For the ABI Prism Big Dye Terminators in a GeneAmp 9600, the recommended regime is 96░C (10 sec), 50░C (5 sec), 60░C (4 min). Different sequencing chemistries and thermal cyclers may require adjustments in the thermal cycling conditions. After 25 rounds of amplification, hold the temperature at 4░C until ready to purify.
  3. Add 2 volumes (10-50 Ál) of Binding Buffer per well. After thermal cycling, remove the 96-well or 384-well plates from the thermal cycler and add 2 volumes (10-50 Ál) of Binding Buffer to each well. The Binding Buffer has a pale green appearance.  Be careful to use Binding Buffer and NOT Wash Buffer, which is clear.
  4. Mix extension products and Binding Buffer by pipetting up and down 10 times. Avoid splashing samples between wells at this step.
  5. Pipette each sample into an individual well of a 96- or 384-well Clean-Up Filter.  Make sure that each sample is transferred correctly from the thermal cycling plate into the Clean-Up Filter, and that the Wash Microplate is positioned underneath the Clean-Up Filter.
  6. Centrifuge for 1 min (500 x g) to remove unbound material. The extension products will remain bound to the filter, and unwanted components such as primers, enzymes, salts and unincorporated dyes and nucleotides will pass through the filter into to Wash Microplate.
  7. Add 80 Ál per well of Wash Buffer and centrifuge for 1 min (500 x g).  This step will remove residual contaminants from the membrane.
  8. Wash the Clean-Up Filter two more times with 80 Ál Wash Buffer.  Use 1 min centrifugation steps to remove Wash Buffer during each cycle.  The Wash Microplate can be used for to collect each round of Wash Buffer. Make certain to empty the Wash Microplate between each wash step.
  9. Centrifuge for 5 min (500 x g) to remove residual Wash Buffer. After the third wash step, spin the Clean-Up Filter containing bound products for 5 min at 500 x g to remove any remaining Wash Buffer from the membrane.  Following centrifugation, discard the Wash Microplate (lower plate) containing the residual Wash Buffer.
  10. Add 50 Ál dH20 to each well of the Clean-Up Filter. Place the Clean-Up Filter containing the purified samples on top of a 96- or 348-well Sample Microplate and add 50 Ál dH20 to each well.  Make sure to pipette the dH20 directly onto the surface of the Clean-Up Filter membrane to allow more efficient wetting.
  11. Incubate Clean-Up Filter for 1 min at room temperature.  This step allows time for the re-suspension of the extension products. Shorter incubation times may reduce yield.
  12. Elute purified products into a 96- or 384-well Sample Microplate by centrifugation for 5 min (500 x g). Place the Sample Microplate beneath the Clean-Up Filter plate, and elute the samples by centrifugation.
  13. Evaporate samples to dryness by vacuum centrifugation (20-40 min). This step removes the dH20 from the extension products, leaving the purified extension products as a dried pellet at the bottom of the well of the Sample Microplate. Use elevated temperatures (e.g. 65░C) to speed up the drying step.
  14. Re-suspend each sample in 3.0 Ál loading buffer and load onto sequencing gel.  Make sure to re-suspend sample completely before loading.  Load the entire 3.0 Ál sample onto a gel and carry out electrophoresis according to the instructions of the manufacturer.

dye-terminator-kits
Figure 3. Clean-Up Filter. The Dye Terminator Clean-Up Kit 384-well (DTC384) Clean-Up Filter shown bottom side up. The unique filter construction and drip directors eliminate glass fiber shedding into samples and well-to-well contamination.  DTC kits should be used for all DNA sequencing and SNP identification applications to improve read length and sequence accuracy.

DNA-sequencing-cleanup
Figure 4. Dye terminator sequencing samples. Samples containing extension products mixed with DTC Binding Buffer can be transferred into a 384-well Clean-Up Filter manually using a pipettor.  The Clean-Up Filter is positioned on top of the Wash Microplate in preparation for centrifugation.  DTC kits should be used for all DNA sequencing and SNP identification applications to improve read length of sequence accuracy.

Equipment Requirements
Thermal Cycler (Perkin Elmer, MJ Research, Stratagene)
Dye terminator kit (ABI)
SpeedVac Centrifuge
Vacuum Manifold (Arrayit)

Troubleshooting Tips
Poor sequence data early in read due to contaminating dyes in purified extension products:

  • Extension products drying on the Clean-Up Filter membrane prior to the wash steps
  • Incomplete washing of extension products bound to Clean-Up Filter membrane

Weak fluorescent signal in sequence traces due to low yield of extension product:

  • Poor amplification due to inferior templates or primers
  • Poor binding of extension products to Clean-Up Filter membrane

Kit Contents
Single column (DTC)

  • Spin Columns (50)
  • Wash Microfuge Tubes (50)
  • Elution Microfuge Tubes (50)
  • Binding Buffer (30 ml)
  • Wash Buffer (30 ml)
  • Handbook (1)

96-well microplate column (DTC96)

  • Clean-Up Filter 96-well (1)
  • Sample Microplate 96-well with cover (1)
  • Wash Microplate 96-well w/o cover (1)
  • Binding Buffer (5 ml)
  • Wash Buffer (30 ml)
  • Handbook (1)

 384-well microplate column (DTC384)

  • Clean-Up Filter 384-well (1)
  • Sample Microplate 384-well with cover (1)
  • Wash Microplate 384-well w/o cover (1)
  • Binding Buffer (25 ml)
  • Wash Buffer (100 ml)
  • Handbook (1)

Ordering Information

Product

Description

Catalog ID

Price per kit (US dollars)*

Dye Terminator Clean-Up Kit, Single Column Version

Single-column dye terminator clean up kit, 50 single tube spin columns, microfuge wash and elution tubes, binding buffer and wash buffer.

DTC

$144

Dye Terminator Clean-Up Kit, 96-Well Version

Microplate-based 96-well purification kit containing buffers, plates and protocols

DTC96

$151

Dye Terminator Clean-Up Kit, 384-Well Version

Microplate-based 384-well purification kit containing buffers, plates and protocols

DTC384

$185

DTC Discounts
1-9 kits, list price
10-49 kits, 15% discount
50-99 kits, 20% discount
100+ kits, 25% discount

DTC96 Discounts
10-49 kits, 25% discount off list price
50-99 kits, 30% discount off list price
100+ kits, 40% discount off list price

DTC384 Discounts
10-49 kits, 20% discount off list price
50-99 kits, 25% discount off list price
100+ kits, 30% discount off list price

*International pricing may vary as much as 30% (or more depending on country) due to import duties, stocking fees and technical support.

*To order ArrayIt® Brand Products: call 408-744-1331, fax 408-744-1711 or click on the purchase buttons above to proceed directly to the purchase page.

Storage Conditions
The ArrayIt® brand Dye Terminator Clean-Up Kits should be stored dry and at room temperature (20-25░C). The kit performs well under a wide range of ambient temperatures and relative humidity. The kits have a shelf life of one-year from the date of purchase.

*Add shipping and handling to all orders.

Warranty
ArrayIt® Brand products have been scientifically developed and are sold for research purposes.  Extreme care and exact attention should be practiced in the use of the materials described herein. All ArrayIt® brand products are subject to extensive quality control and are guaranteed to perform as described when used properly. Any problems with any ArrayIt® brand product should be reported to Arrayit immediately. Arrayit’s liability is limited to the replacement of the product, or a full refund.  Any misuse of this product including deviations from our protocols is the full responsibility of the user, and Arrayit makes no warranty or guarantee under such circumstances.

 

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