DNAstable® Plus Handbook:
Preserve and store DNA samples at room temperature

Protocols for Sample Protection, Sample Recovery and Downstream Applications

Introduction

Biomatrica offers innovative technologies for stabilizing biological samples at room temperature. We have developed a novel platform technology designed for use in protecting complex biological samples and assays. Sample preservation is achieved without degradation, thus enabling laboratories to decrease their reliance on freezers and drastically reduce shipping costs.

DNAstable Plus is a unique storage medium that preserves purified genomic DNA, at room temperature, in liquid format for up to one year, and as a drydown for longer storage periods. Dried DNAstable Plus allows for long-term stabilization of DNA samples with easy sample recovery by simply adding water.

Stabilize and store purified DNA at Room Temperature.

DNAstable Plus enables you to preserve, store and ship your purified DNA samples at room temperature in liquid and dry state. It offers workflow flexibility with thermostability at even extreme temperatures and ability to handle high concentrations of DNA. Purified DNA is preserved in liquid or dry-down state. Recovery from dry matrix is achieved within minutes by simply adding water. Sample is ready for any downstream analysis without need for any additional purification step.

DNA protection is achieved during processing and storage throughout the workflow:

  1. Liquid Stabilization (short and medium-term)
  2. Liquid-to-Dry (LD) Stabilization (long-term)

Using DNAstable Plus, you can completely recover your dry DNA samples by simple rehydration - just add water. In addition, you can concentrate your sample during recovery. Samples stored in liquid format or rehydrated are ready for immediate use, without the need for further purification. Downstream applications using recovered samples include:

  • Restriction enzyme analysis
  • Sequencing
  • PCR
  • Quantitative PCR
  • SNP genotyping

Principles and Procedures

DNAstable Plus is a mixture of dissolvable stabilizers that protect DNA from degradation. Based on the unique thermo-stable properties of Biomatrica’s proprietary formulation, DNAstable Plus forms a protective seal around DNA as it dries, effectively “shrink-wrapping” the sample in a protective coating. Drying can occur at ambient temperatures with a vacuum concentrator (i.e. SpeedVac®) or through passive air drying. Stored dry at ambient temperatures, the protected DNA can be safely stored for extended time periods. Currently, Biomatrica has accelerated aging studies indicate that DNA can be safely stored for up to 15 years at room temperature. Please note that we recommend storing dry samples in either a dry storage cabinet or a heat-sealed, moisture-barrier bag. Samples can be recovered through simple rehydration and are ready for immediate use, without the need for further purification.

Storage

DNAstable Plus reagent can be stored at 4°C to 20°C until ready for use. The shelf-life for the reagent is indicated on the label. As an example, Best use before 04/12 corresponds to the last date of April 2012.

Extra Sample Pouches/Desiccant Packs are sold as sets of twelve, catalog no. 14001-007. The sample pouch allows for dry storage even in uncontrolled humidity environments and serves as an alternative to storage in a dry storage cabinet. The moisturebarrier foil bag should be opened just before use.

Samples protected for Liquid-to-Dry (LD) storage should be inserted along with a desiccant packet into the pouch and heat-sealed. Alternatively, store dried samples in a dry storage cabinet at room temperature (15-25°C).

For optimal results when shipping dry samples protected in DNAstable Plus, please utilize moisture barrier foil bag (sample pouch) along with a desiccant packet.

Applications

Sample Type

DNAstable Plus has been used for room temperature dry storage of genomic DNA.

Assay Type

DNA stored in DNAstable Plus is ready for immediate use in the following applications:

  • SNP analysis
  • Restriction enzyme analysis
  • Agarose-gel Electrophoresis
  • Next-Generation Sequencing
  • PCR
  • Quantitative PCR

Shipping

DNAstable Plus provides the ideal format for transport and shipping of samples at ambient temperatures. Individual tubes or bottles can be shipped conveniently at room temperature, without the need for cold packs, dry ice or Styrofoam® packing, thus greatly reducing shipping costs. Fluctuating temperatures or delays during transport do not affect samples protected in DNAstable Plus. (Additional Sample Pouches/Desiccant Packs are available for purchase as sets of 10, catalog no. 14001-007.)

Important Notes

Please take a few moments to read this handbook carefully before beginning the sample stabilization step for dry storage of DNA at room temperature. For optimal protection and sample recovery, DNA should be purified of any contaminating DNase activity.

Sample Application and Drying

To store DNA samples in liquid format, just add the 5X DNAstable Plus buffer and store at room temperature. Samples to be stored in dry format in DNAstable Plus must be dried completely for optimal protection and stability during room temperature storage. Sample volumes of up to 50 µl can be applied directly to DNAstable Plus and dried at ambient temperatures on the lab bench or in a laminar flow hood (recommended). Larger sample volumes may require use of a vacuum concentrator for complete drying.

For optimal results, store between 1 ng and 250 µg of total DNA per tube in liquid format, or up to 50ug in dry format.

Sample Storage

Store liquid samples at room temperature in closed tubes, to prevent evaporation. Store dried samples in either (a) a dry storage cabinet at room temperature (15-25°C or 59-77°F) or (b) a heat-sealed, moisture-barrier bag along with a silica gel desiccant pack.

Sample Recovery

To recover samples stored in dry format in DNAstable Plus, just add water. Samples are ready for immediate use in downstream applications. It is not necessary to further purify rehydrated samples. Aqueous solutions such as TE buffer (10 mM Tris HCl, 1 mM EDTA), PCR reaction buffers, and restriction enzyme buffers are also compatible with the recovery of samples from DNAstable Plus.

Protocol: Sample preparation

This protocol is designed for the preparation of purified genomic DNA for storage in DNAstable Plus.

Purification Techniques

Most standard molecular biology techniques and/or commercially available kits are compatible with DNAstable Plus storage. For optimal results, DNA samples should be free of any contaminating DNase activity.

Purified DNA that is DNase-free and resuspended in water or a low salt buffer can be used directly with DNAstable Plus.

Determining yield

The concentration of the DNA sample should be determined prior to sample application into DNAstable Plus. Although not essential, applying a known amount of DNA into DNAstable Plus for storage can facilitate sample retrieval and subsequent applications.

For optimal results, store between 1 ng and 250 µg of total DNA per tube in liquid format, or up to 50 µg in dry format.

Protocol: Sample drying and storage

DNAstable Plus preserves DNA samples at room temperature. Each tube or bottle contains DNAstable Plus provided as 5X buffer solution which protects nanogram to microgram amounts of DNA. The sample can then be stored in liquid for up to a year at room temperature. For longer term storage, the sample must be completely dried for maximum protection and stability for storage at ambient temperatures.

Note: In the event you want to determine the DNA concentration at a later date using a spectrophotometer, keep an unused amount of DNAstable Plus to generate a blank. See Appendix A.

Procedure

  1. Add 25% (vol:vol) of the reagent to the DNA sample (≤250µg), stored in water or any aqueous buffer (e.g., 25 µl of DNAstable Plus to 100 µl of DNA).
  2. Mix the sample thoroughly with gentle pipetting. Avoid forming air bubbles.
  3. Store at room temperature for up to a year, or proceed to 4.
  4. Dry the uncovered sample completely at room temperature (15-25°C). We recommend using a laminar flow hood or drying under a vacuum concentrator to ensure complete drying. Recommended drying times are given in Tables 1 and 2.

    Note: Drying should occur at ambient laboratory temperatures (15°C–25°C). Exposure to moisture for extended periods of time will reduce product performance and sample protection. Climate-controlled laboratory environments and buildings are normally maintained at 40-50% relative humidity levels. We recommend drying under a vacuum concentrator if conditions exceed these parameters.

    Table 1. Minimal Drying Times in a Laminar Flow Hood.*
    Volume (µl) Drying Times (hrs)
    Tubes
    Drying Times (hrs)
    (96-well plate)
    Drying Times (hrs)
    (384-well plate)
    5 4 4 8
    6-10 6 6 12
    11-20 12 8 24
    21-50 28 18 48
    51-100 56 24 68
    101-125 72 24 78

    *Drying time may vary dependent on the humidity level in the laboratory. Recommended drying times were determined at 50% relative humidity (RH); (typical HVAC controlled facilities have 40-50% RH). Volume and Drying times refer to the mixture of sample and DNAstable Plus reagent.

    Longer drying times are preferable to ensure complete drying. Completely dried samples should NOT feel sticky or tacky when tapped with a sterile pipette tip.

    Table 2. Minimal Drying Times in a Vacuum Concentrator at Low Temperature (25-30°C)**
    Volume (µl) Drying Times (Min)
    Tubes
    Drying Times (Min)
    (96-well plate)
    Drying Times (Min)
    (384-well plate)
    5 10 15 80
    6-10 15 15 120
    11-20 30 30 180
    21-50 45 90 360
    51-100 60 150
    101-125 75 180
    ** Drying times may vary depending on model and condition of vacuum concentrator used. Volume and Drying times refer to the mixture of sample and DNAstable Plus reagent.

    Note: Do not exceed 50 µg of DNA per well or tube to ensure optimal protection.
  5. Cover samples after drying and store at room temperature, protected from moisture.

    After complete sample drying, plates should be re-sealed with aluminum foil seals. Tubes can be closed using the cap supplied with the tube.

Protocol: Drydown Sample recovery

DNA stored dry in DNAstable Plus can be recovered by the addition of water or aqueous buffer. Samples are ready for downstream applications without the need for further purification.

Procedure

  1. Add water or aqueous buffer directly to the dried sample in DNAstable Plus tube or well, to the same volume as dried down sample.

    Individual wells in a plate can be opened by puncturing the aluminum foil seal with a pipette tip or razor blade.

    Samples may be rehydrated directly with aqueous buffers used for downstream applications such as restriction enzyme buffers, PCR buffers, etc.

    Note: Samples can be easily concentrated at this step if the amount of water or buffer used to rehydrate a known amount of dried DNA is smaller than the original sample. However, downstream applications might be affected by this concentration.

  2. Incubate at room temperature for 15 minutes to allow complete rehydration.
  3. Mix the sample by gently pipetting up and down to resuspend the sample. Avoid forming bubbles while pipetting.

    The rehydrated sample is now ready for use in downstream applications. Real-time PCR analysis using SYBR® Green or sequence-specific probes should be performed using the guidelines as provided in Appendix B.

    To determine DNA concentration and recovery yield, see Appendix A for directions on how to perform UV spectrophotometry (i.e. A260 reading) using samples stored in DNAstable Plus. Spectrophotometer must be zeroed against a DNAstable Plus blank.

    Store unused rehydrated samples at 4°C or room temperature for medium term storage (up to 1 month).
    Rehydrated sample contain DNAstable Plus and can be re-dried without lost of efficient sample stabilization. We do not recommend repeating this process more than 5 times.

Protocol: Downstream applications

Rehydrated DNAstable Plus samples do not require further purification for the majority of molecular biology applications, and can be used directly without interference or inhibition in downstream applications. Go here for complete list of suitable applications. For further results and examples, refer to www.biomatrica.com under Application Notes.

Real-time PCR analysis using SYBR® Green or sequence-specific probes should be performed using the guidelines as provided in Appendix B.

If necessary, samples can be purified using column purification technology from commercially available kits.

Troubleshooting Guide

Dry storage of DNA in DNAstable Plus is extremely effective for storage of samples at room temperature, if attention is paid to ensure complete drying and proper storage conditions (i.e. between 15-25°C and protected from moisture). Stabilized samples should be placed in either (a) a dry storage cabinet at room temperature (15-25°C) or (b) a heat-sealed, moisture-barrier bag to ensure a permanently controlled environment. Significant moisture content in the air will hydrate DNAstable Plus, resulting in sample degradation.

The following troubleshooting guide may be helpful in solving any problems that arise. Scientists at Biomatrica’s Technical Service Department are available to answer questions about the information and protocols in this handbook or general molecular biology applications.

Situation Comment Suggextion
DNases in sample DNA to be stored in DNAstable Plus must be free of contaminating DNase activity for optimal protection. Ensure that samples are purified so that they are DNase-free before applying to DNAstable Plus.
Low amount of DNA in initial sample Before applying the sample, measure the concentration and record the amount of DNA added. Identical samples can be rehydrated separately.
Sample not properly applied Sample must be diluted into 5X DNAstable Plus buffer, to ensure stabilization. Add 4 parts of DNA solution to 1 part of DNAstable Plus buffer and store at room temperature. For long term storage, dry sample and store at room temperature.
Too much buffer used for rehydration Use of large volumes (>100 µl) for rehydration may cause overflow of wells and crosscontamination between wells. Samples can be rehydrated in the recommended volumes and then transferred to a larger vessel and brought up to a larger volume.
Less volume is recovered from well than was initially added for rehydration Rehydration of the dissolvable matrix may cause a reduction in sample volume recovery. This is especially noticeable for smaller volumes (i.e. <20 µl). — Minimal loss of recovered sample volume does not affect the stability or performance of DNA or DNAstable Plus in downstream applications.
— A 10% loss in volume can be included for adjustment (e.g. add 110 µl of water to ensure recovery of 100 µl of rehydrated sample).

Appendix A: UV spectrophotometry with DNAstable Plus

The absorbance of UV light (260 nm) can be used to determine the concentration of rehydrated DNAstable Plus samples.

Procedure

  1. Prepare sample and blank

    The UV spectrophotometer must be calibrated against an aliquot of DNAstable Plus for accurate A260 readings.

    Sample: Rehydrate well or tube containing sample stored in DNAstable Plus in a final volume large enough to obtain an accurate absorbance reading (volume is dependent on cuvette size). The absorbance will be accurate up to a reading of 2, at which point sample dilution may be necessary to obtain an accurate A260 reading.

    Blank: Calibrate the spectrophotometer against an aliquot of DNAstable Plus for use as the reference sample. Reference the spectrophotometer against DNAstable Plus.

    Calibrate the spectrophotometer using the reference sample.
  2. Determine A260 reading of sample.
  3. Calculate concentration of sample.

    The nucleic acid concentration for a standard cuvette with 1 cm path length can be calculated as follows:

    For double-stranded nucleic acid
         Conc. (µg/ml) = (A260 reading) (50 µg/ml) (dilution factor)

    For single-stranded nucleic acid
         Conc. (µg/ml) = (A260 reading) (38 µg/ml) (dilution factor)

Appendix B: Quantitative PCR using samples stored in DNAstable Plus

To quantify samples using Picogreen

Add DNAstable Plus directly to the Picogreen standards. For example, if you recovered your DNA sample stored in DNAstable Plus in 20 µl and you are quantifying 2 µl of sample, add 2 µl of DNAstable Plus to 18 µl of Picogreen standards.

Using TaqMan®

A 1:5 final dilution of sample:PCR Master Mix total final volume is recommended. Samples should be rehydrated in a volume appropriate for up to 50 µl QPCR reaction (total final volume) prior to following manufacturer’s instructions.

Example: TaqMan® PCR Master Mix Reagents Kit

  1. Human genomic DNA (Novagen; Madison, WI) was diluted to 0.2 ng/µl in water or 1X DNAstable Plus matrix and 10 µl dried down in a safety hood.
  2. Samples were later rehydrated with 10 µl of water as described in “Recovering DNAstable Plus Samples” steps 1-5 above.
  3. For each QPCR reaction, the entire 10 µl of rehydrated sample was added to 40 µl of QPCR master mix containing:
    25.00 µl TaqMan Universal PCR Master Mix (Applied Biosystems)
    1.25 µl Probe
    1.00 µl 10 µM forward primer
    1.00 µl 10 µM reverse primer
    11.75 µl Water
    10.00 µl Rehydrated DNA sample
    50.00 µl Total Final Volume
  4. A Thermal Cycler (ABI 7300 Real Time PCR System) was used with the following conditions:
    Initial step:
    50°C for 2 min
    95°C for 10 min
    Remaining steps:
    40 cycles each
    95°C for 15 sec
    60°C for 1 min

Results from Example: TaqMan® PCR Master Mix Reagents Kit

The concentration of DNA as determined by QPCR was found to be equivalent between test and control samples (±10%). A slope of -3.1 to -3.6 and an R2 value of 0.99 are considered acceptable.

SYBR® Green Technology

A dilution of sample to PCR total volume is recommended for DNAstable Plus samples used in QPCR with SYBR Green reagents. Up to 30% of the QPCR reaction can be composed of the 1X DNAstable Plus matrix.

Example SYBR® Green reaction

  1. Human genomic DNA (Novagen; Madison, WI) was diluted to 1X DNAstable Plus formulation and then dried down in a safety hood.
  2. Samples were rehydrated with 20 µl of water as described in “Recovering DNAstable Plus Samples” steps 1-5 above.
  3. For each QPCR reaction, 7.5 µl of rehydrated sample was added to 17.5 µl of QPCR master mix following manufacturer’s instructions, to a final QPCR reaction volume of 25 µl.
  4. Use thermal cycling reaction conditions as recommended by the manufacturer.

Quality Control

Every manufacturing production lot of DNAstable Plus is quality control tested for contamination and functional performance. All products are tested against predetermined specifications to ensure consistent product quality.

Product Use Limitations

DNAstable Plus technology is specifically designed for the stabilization of purified DNA samples. No claim or representation is intended for their use on any other biological materials. DNAstable Plus products are For Research Use Only and are not intended for use in diagnostic procedures. For optimal performance, products must be used and stored according to manufacturer’s guidelines.

Optimal protection of DNA in DNAstable Plus occurs when samples are prepared and stored at room temperature and, for dry storage, in a relative humidity environment ≤ 40%. Please refer to “Sample drying and storage” for details on preparing and storing DNA under relative humidity conditions exceeding 40%.

Ordering Information

Product Name Catalog No. Description
DNAstable Plus 2ml 53091-016 (1) 2ml tube of liquid DNAstable Plus for liquid and Liquid-to-Dry (LD) storage of DNA
DNAstable Plus 10ml 52091-026 (1) 10ml bottle of liquid DNAstable Plus for liquid and Liquid-to-Dry (LD) storage of DNA