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Products - DNA Purification - PCR Purification - PCR 96-Well 100 for Rapid and Efficient Microplate Purification of PCR Products for Microarrays and DNA Sequencing

microarray-PCR
Arrayit’s PCR Purification Kits allow high-throughput purification of PCR products for DNA microarrays, DNA sequencing, and other applications in genomics.

Table of Contents

  • Introduction
  • Quality Control
  • Product Description
  • Technical Notes
  • Technical Assistance
  • Short Protocol
  • Complete Protocol
  • Equipment and Reagents
  • Troubleshooting Tips
  • Kit Contents
  • Scientific Publications
  • 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 including the steps and principles needed to use Arrayit’s ArrayIt® brand 96-well PCR Purification Kit (PCR96-100) for 100 Ál PCR samples.

Quality Control
Arrayit assures the performance of these products.  The finest scientific research went into the development of these products. 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
Arrayit’s ArrayIt® brand PCR Purification Kits are advanced purification systems that use sophisticated membrane separation technology in both 96-well and 384-well formats. PCR Purification Kits increase the quality of DNA microarray data by removing unwanted salts, enzymes, primers, unincorporated nucleotides, and other contaminants from amplified products that diminish the quality of DNA hybridization reactions and elevate fluorescent background.

Users will appreciate the following features:

  1. Improves spot signal intensities
  2. Reduces spot background
  3. Increases coupling efficiency
  4. Improves sample-to-sample printing consistency
  5. Supports native and modified PCR products
  6. Supports two- and three-dimensional microarray surfaces
  7. Superior SuperFilter separation chemistry provides >99% PCR purity
  8. >90% yield compared to 25-75% for other kits
  9. Purifies 50-10,000 bp PCR products
  10. Much better than ethanol precipitation
  11. No glass fiber shedding like other PCR Purification Kits
  12. Kits arrive ready to use, no buffer or column preparation required
  13. All buffers sterile-filtered for optimal performance
  14. Ultra-pure reagents used for superior performance
  15. Can be used manually or with automated liquid handling devices
  16. 96- and 384-well microplate formats provides high throughput

Technical Notes
Arrayit’s ArrayIt® 96-well 96100 PCR Purification Kit is designed for 100 Ál PCR samples, but can also be used for smaller sample volumes ranging from 25-100 Ál. For PCR samples smaller than 100 Ál, simply scale down the volume of Binding Buffer proportionately. Samples can be eluted using 1X TE buffer (see Step 9), or distilled H2O in cases where Tris or EDTA may inhibit downstream reactions.

microarray-PCR-reaction
Figure 1.  Gel electrophoresis products purified with Arrayit’s 96-well PCR Purification Kit (PCR96100).  A 10 Ál sample of each 100 Ál reaction was separated on 0.8% agarose, stained with ethidium bromide, and the data were coded to a rainbow palette. Intense equimolar products bands are clearly visible.

cDNA-microarray
Figure 2.  Microarray signal intensities generated with cDNA microarrays printed from PCR products purified by ethanol precipitation (Ethanol) or with Arrayit’s 96-well PCR Purification Kit (Arrayit). Microarrays were hybridized and scanned and the fluorescent data were coded to a rainbow palette.  Stronger signals with PCR products purified with Arrayit’s PCR96100 Kit are easily observed. Data taken from Hedge et al., BioTechniques.

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!

Short Protocol (96-well for 100 Ál PCR samples)

  1. Obtain 100 Ál PCR samples.
  2. Add 5 volumes (500 Ál) of Binding Buffer to the SuperFilter800.
  3. Quickly add the 100 Ál PCR samples to the SuperFilter800.
  4. Mix thoroughly by pipetting.
  5. Pass samples through SuperFilter800 by vacuum filtration.
  6. Wash one time with 800 Ál Wash Buffer.
  7. Wash two times with 100 Ál Wash Buffer.
  8. Dry SuperFilter800 membrane completely by vacuum or centrifugation.
  9. Add 100 Ál 0.1X TE (8.0).
  10. Incubate 5 min.
  11. Elute the purified PCR products into the 96-Well Marked Microplate.
  12. Dry the PCR products completely by vacuum centrifugation.
  13. Re-suspend in 10-20 Ál Printing Solution.
  14. Print the purified PCR products onto Microarray Substrates.

Complete Protocol (96-well for 100 Ál PCR samples)

  1. Obtain 100 Ál PCR reactions. Set up any polymerase chain reaction (PCR) of choice in a 100 Ál volume.  Standard PCR reactions contain 1X amplification buffer (50 mM KCl, 10 mM Tris-Cl pH 8.3, 1.5 mM MgCl2, 0.01% gelatin), 200 ÁM each of the four dNTPs, 100 pmoles each primer, 20-200 ng genomic DNA, and 2.5 units of Taq DNA polymerase.  A standard PCR cycling regime is 30 cycles at three temperatures (94░C, 50░C and 72░C), with 30 sec at each temperature.  The PCR reactions can be run in a 96-well format and stored at 4░C or –20░C prior to purification.
  2. Add 5 volumes (500 Ál) of Binding Buffer to the SuperFilter800.  Place the SuperFilter800 on a vacuum manifold and add 500 Ál Binding Buffer to each well using an automatic pipette or liquid handling robot. This step should be performed within 60 sec to avoid loss of Binding Buffer, which passes slowly through the SuperFilter800 membrane by gravity flow.
  3. Quickly add the 100 Ál PCR reactions to the SuperFilter800.  To the 500 Ál of Binding Buffer in each well, add the 100 Ál PCR samples. Be careful not to splash the samples from well to well. This step can be performed manually by pipetting, or with an automated liquid handling device.
  4. Mix thoroughly by pipetting. Using a pipette or automated liquid handling device, mix the 500 Ál Binding Buffer and 100 Ál PCR samples by pipetting up and down at least 10 times.  It is essential to mix the samples completely at this step, and failure to do so may reduce yield.
  5. Pass samples through SuperFilter800 by vacuum filtration.  The mixed 600 Ál samples should be passed slowly through the SuperFilter800 using a mild vacuum.  The filtration process should take 30-60 seconds for efficient binding of the PCR products to the SuperFilter800 membrane. This step can also be performed by centrifugation for 60 sec at 500 x g.  For the centrifugation protocol, fit the SuperFilter directly into the 96-Well Unmarked Microplate and pre-spin briefly to collect half of the sample volume (300 Ál).  Empty the Unmarked Microplate, then spin for 60 sec at 500 x g to filter the remaining 300 Ál of sample. At the end of the centrifugation step, discard the eluent by inverting the Unmarked Microplate.
  6. Wash one time with 800 Ál Wash Buffer. After the 600 Ál sample is filtered completely through the SuperFilter800, wash the SuperFilter800 with 800 Ál of Wash Buffer.  This wash will remove sample adhering to the walls of the SuperFilter 800, as well as enzymes, salts, buffer components, nucleotides and primers from the SuperFilter membrane.  Mild vacuum filtration or centrifugation for 60 sec at 500 x g can be used for this wash step.  Do not allow unwashed samples to dry on the SuperFilter membrane prior to the wash steps, as this will lead to contamination of the PCR products.
  7. Wash two times with 100 Ál Wash Buffer.  After the 800 Ál wash step, wash the SuperFilter800 membrane with two additional washes of 100 Ál each. These washes will remove any residual contaminants. Mild vacuum filtration or centrifugation for 60 sec at 500 x g can be used for these wash steps.
  8. Dry SuperFilter800 membrane completely by vacuum or centrifugation.  This step removes residual Wash Buffer remaining in the SuperFilter800 that can reduce yield.  A 5 min vacuum treatment under strong vacuum or centrifugation for 5 min at 500 x g both work well for the drying step.  For the centrifugation protocol, the 96-Well Unmarked Microplate should be used to catch the eluent, and discarded after the drying step.
  9. Add 100 Ál 0.1X TE (8.0).  Place the SuperFilter800 containing the bound PCR products onto a 96-Well Marked Microplate and add 100 Ál of 0.1X TE pH 8.0 to each well. Pipette the 0.1X TE directly onto the membrane containing the bound PCR product. Do not use 1X TE as the EDTA in the concentrated sample may inhibit coupling to microarray substrates.
  10. Incubate 5 min.  This step allows the bound PCR product to re-hydrate prior to the elution step.  Failure to wait 5 min prior to elution may reduce yield.
  11. Elute into 96-Well Marked Microplate.  Centrifuge the two plates for 5 min at 500 x g to elute the purified PCR products into the 96-Well Marked Microplate. The volume of the eluted sample will be ~80 Ál.
  12. Dry samples completely by vacuum centrigation. This step removes dH20 from the sample, leaving dry DNA pellets at the bottom of each well in the 96-Well Marked Microplate.  A 30 minute vacuum centrifugation at 42-65░C should be sufficient to dry the samples completely, though additional drying time can be used without damaging the samples.
  13. Re-suspend in 10-20 Ál Printing Solution.  The dried and purified PCR products should be re-suspended in an appropriate printing solution such as Arrayit’s MicroSpotting Solution Plus.  The yield from a typical 100 Ál PCR sample will be 3-10 Ág, producing a final DNA concentration of 0.15-1.0 Ág/Ál.
  14. Print purified PCR products onto Microarray Substrates.  High-quality microarray substrates such as SuperAmine, SuperAldehyde, SuperEpoxy, MirrorAmine, MirroAldeyde, MirrorEpoxy should be used to obtain the best results.  Users wishing to make their own substrates should use SuperClean or MirrorClean for best results. Optically flat substrates manufactured in a cleanroom environment consistently produce uniform spots, strong signal intensities and low and uniform background.

Equipment and Reagents
Microplate Vacuum Manifold
Microarray Slides
Microarray Super Substrates
Microarray Hybridization Buffers
Hybridization Cassettes
Cleanroom Wipes

Troubleshooting Tips
Purified PCR products contain contaminating primers

  • May actually be primer dimers (small double-stranded products)
  • Use less primer (≤100 pmoles per 100 Ál reaction)
  • Do not allow Binding Buffer dry on SuperFilter membrane
  • Reduce Binding Buffer from 5 volumes to 3 volumes

Low yield

  • Check efficiency of PCR reaction
  • Poor elution due to residual Wash Buffer in the SuperFilter800

cDNA-microarrays
Figure 3.  96-Well SuperFilter800 containing 48 samples of 600 Ál of Binding Buffer mixed with 100 Ál PCR samples. The SuperFilter800 is positioned on a 96-Well Unmarked Microplate in preparation for the filtration step.

Kit Contents (96-well for 100 Ál PCR reactions)

  • 96-Well SuperFilter800 (1)
  • 96-Well Unmarked Microplate (1)
  • 96-Well Marked Microplate and Cover (1)
  • Binding Buffer (75 ml)
  • Wash Buffer (150 ml)
  • Handbook (1)

96-well-PCR-plate
Figure 4.  The sample drip directors on the underside of a 96-Well SuperFilter800 are constructed to maximize sample recovery and eliminate well-to-well contamination.

Scientific Publications
Arrayit PCR Purification Kits are featured in hundreds of scientific publications.

Ordering Information:

Product

Description

Catalog ID

Price (US dollars)*

PCR Purification Kit, 96-well for 100 Ál PCR reactions

96-well PCR purification kit containing SuperFilter800, Microplates, buffers, and protocols

PCR96100

$174

PCR96100 discounts:
10-49 kits, 25% discount
50-99 kits, 30% discount
100+ kits, 45% discount

*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 button to proceed directly to the purchase page.

Storage Conditions
Arrayit’s ArrayIt® brand PCR Purification Kits should be stored dry at room temperature (20-25░C). The kits perform well across a wide range of ambient temperatures and relative humidity. The kits are nuclease-free, sterile, and have a shelf life of one-year from the date of purchase.

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 our 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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