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Products - DNA Purification - PCR Purification - PCR 96-Well 50 Kits for Plate Purification of 50 Ál PCR Reactions for DNA Microarrays and DNA Sequencing

PCR-reactions
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 (PCR9650) for 50 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:

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

Technical Notes
Arrayit’s ArrayIt® 96-well 9650 PCR Purification Kit is designed for 50 Ál PCR samples, but can also be used for smaller sample volumes ranging from 10-50 Ál. For PCR samples smaller than 50 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.

PCR-product
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.  Similar results have been obtained with the PCR9650 Kit.

PCR-products
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, and similar results have been obtained with the PCR9650 Kit. Data provided courtsey of Hegde et al., BioTechniques 29, 548-550, 2000.

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 50 Ál PCR samples)

  1. Obtain 50 Ál PCR samples.
  2. Add 4 volumes (200 Ál) of Binding Buffer to the SuperFilter350.
  3. Quickly add the 50 Ál PCR samples to the SuperFilter350.
  4. Mix thoroughly by pipetting.
  5. Pass samples through SuperFilter350 by vacuum filtration.
  6. Wash 3 times with 350 Ál Wash Buffer.
  7. Dry SuperFilter350 membrane completely by vacuum or centrifugation.
  8. Add 100 Ál 0.1X TE (8.0) and incubate 5 min.
  9. Elute the purified PCR products into the 96-Well Marked Microplate.
  10. Dry the PCR products completely by vacuum centrifugation.
  11. Re-suspend in 2.5-10 Ál Printing Solution.
  12. Print the purified PCR products onto Microarray Substrates.

Complete Protocol (96-well for 50 Ál PCR samples)

  1. Obtain 50 Ál PCR samples.  Set up any polymerase chain reaction (PCR) of choice in a 50 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, 50 pmoles each primer, 5-50 ng genomic DNA, and 1.0 unit 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 4 volumes (200 Ál) of Binding Buffer to the SuperFilter350.  Place the SuperFilter350 on a vacuum manifold and add 200 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 SuperFilter350 membrane by gravity flow.
  3. Quickly add the 50 Ál PCR samples to the SuperFilter350. To the 200 Ál of Binding Buffer in each well, add the 50 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 200 Ál Binding Buffer and 50 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 SuperFilter350 by vacuum filtration.  The mixed 250 Ál samples should be passed slowly through the SuperFilter350 using a mild vacuum.  The filtration process should take 30-60 seconds for efficient binding of the PCR products to the SuperFilter350 membrane. This step can also be performed by centrifugation for 60 sec at 500 x g.  For the centrifugation protocol, fit the SuperFilter350 directly into the 96-Well Unmarked Microplate and spin for 60 sec at 500 x g. At the end of the centrifugation step, discard the eluent by inverting the 96-Well Unmarked Microplate.
  6. Wash 3 times with 350 Ál Wash Buffer.  After the 250 Ál sample is filtered completely through the SuperFilter350, wash the SuperFilter350 with 3 times with 350 Ál of Wash Buffer. The wash steps will remove sample adhering to the walls of the SuperFilter350, 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 into the 96-Well Unmarked Microplate can be used for the wash steps. For the centrifugation protocol, make certain to empty the 96-Well Unmarked Microplate after each centrifugation step.  Do not allow unwashed samples to dry on the SuperFilter350 membrane prior to the wash steps, as this will lead to contamination of the PCR products.
  7. Dry SuperFilter350 membrane completely by vacuum or centrifugation.  This step removes residual Wash Buffer remaining in the SuperFilter350 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.
  8. Add 100 Ál 0.1X TE (8.0) and incubate 5 min. Place the SuperFilter350 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. After pipetting the 0.1X TE, wait 5 min before proceeding to the elution step. This step allows the bound PCR product to re-hydrate, and failure to wait 5 min prior to elution may reduce yield.
  9. Elute the purified PCR products into the 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.
  10. Dry the PCR products completely by vacuum centrifugation.  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 may be used without damaging the samples.
  11. Re-suspend in 2.5-10 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 50 Ál PCR sample will be 1.0-2.5 Ág, producing a final DNA concentration of 0.1-1.0 Ág/Ál.
  12. Print the 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
Vacuum Filter Manifold
Super Microarray Substrates
Mirror Substrates
Hybridization Solutions
Hybridization Cassettes
Cleanroom Wipes

Troubleshooting Tips
Purified PCR products contain contaminating primers

  • May actually be primer dimers (small double-stranded products)
  • Use less primer (≤50 pmoles per 50 Ál reaction)
  • Do not allow Binding Buffer dry on SuperFilter membrane
  • Reduce Binding Buffer from 4 volumes to 3 volumes

Low yield

  • Check efficiency of PCR reaction
  • Poor elution due to residual Wash Buffer in the SuperFilter350

PCR-microarray
Figure 3.  96-Well SuperFilter350 containing 96 samples of 200 Ál of Binding Buffer mixed with 50 Ál PCR samples.  The SuperFilter350 is positioned on a 96-Well Unmarked Microplate in preparation for the filtration step. Samples can be pipetted manually using a multi-channel pipetting device.  The PCR9650 produces 90% yield and >99% sample purity.  PCR purification is strongly recommended for all cDNA microarray applications.

Kit Contents (96-well for 50 Ál PCR reactions)

  • 96-Well SuperFilter350 (1)
  • 96-Well Unmarked Microplate (1)
  • 96-Well Marked Microplate and Cover (1)
  • Binding Buffer (25 ml)
  • Wash Buffer (150 ml)
  • Handbook (1)

PCR_plates
Figure 4.  Shown is the SuperFilter350 containing 96 samples of 250 Ál each, positioned onto a 96-well Unmarked Microplate. Sample filtration is performed by centrifugation for 5 min at 500 x g.  The PCR9650 produces 90% yield and >99% sample purity.  PCR purification is strongly recommended for all cDNA microarray applications.

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 50 Ál PCR reactions

96-well PCR purification kit containing SuperFilter350, Microplates, buffers, and protocols

PCR9650

$151

PCR9650 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 only. ArrayIt® brand products are not for use in human diagnostics or for drug purposes, nor are they to be administered to humans in any way.  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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