CloneStable® Protocol

Product Nos. 93121-017 and 90121-007

Product Description

CloneStable is a unique storage medium that preserves genomic and plasmid DNA at room temperature. CloneStable allows for long-term stabilization of DNA samples with easy sample recovery by simply adding water. Bacterial cells do not survive more than four weeks after dry storage in CloneStable, but bacterial genomic and plasmid DNA are preserved and protected from degradation. Rehydrated samples are ready for immediate use; for most applications, no further purification is needed.

CloneStable is supplied as a coating on the bottom of each well or tube. The aqueous sample will rehydrate the CloneStable within minutes and form a protective barrier around the bacterial DNA as it dries.

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Procedures

Storage of DNA in Bacterial Suspension

Aliquots of E. coli growth cultures or suspensions can be applied directly into CloneStable for storage of total bacterial DNA. The bacteria should be added directly into CloneStable as a liquid suspension to facilitate contact between the sample and the protective matrix. The growth medium (i.e. LB medium) and selective antibiotics (e.g. ampicillin, kanamycin) do not interfere with the storage capacity of CloneStable so there is no need to purify or wash the bacterial suspension. Note: Glycerol stocks should not be added directly to CloneStable. See the detailed protocol on transfer of glycerol stocks for storage in CloneStable.

  1. Calculate the amount of E. coli or total DNA to be stored in each well or tube containing CloneStable. Each well or tube of CloneStable can accommodate E. coli bacterial growth cultures in log or stationary phase with cell densities as high as OD600 = 4.0. We recommend spotting 20 µl of a culture with OD600 = 2.0 (~2x109 cells /ml) onto CloneStable. This gives a final cell density of ~4x107 cells per well.
  2. Heat Lysis (optional). Bacteria are viable in CloneStable in a dried state for up to a month. If the sample will be used for transformation into a new strain within the first month of storage heat the bacterial suspension at 80°C for 5 minutes prior to application to ConeStable to completely lyse the cells.
  3. Gently apply the sample into the center of the CloneStable vessel. Allow the matrix to be hydrated by the aqueous sample for 5-10 minutes. The final volume of the sample to be applied to each well should be ≤ 20 µl.
  4. Mix the sample thoroughly with gentle pipetting (avoid forming air bubbles).
  5. Dry the uncovered sample completely at room temperature. We recommend using a laminar flow hood (see table 1 below for drying times) or drying with a vacuum concentrator (see table 2 below for drying times).

Table 1 – drying times using a laminar flow hood

Sample Volume (μl) Drying Times (hrs)
1–5 2
5–10 6
10–20 10–12

 

Table 2 – drying times using a vacuum concentrator

Sample Volume (μl) Drying Times (hrs)
10–20 0.5

 

  1. Cover samples and store at room temperature. After complete sample drying, plates should be re-sealed with aluminum foil seals (provided in kit) to protect the sample. Tubes can be closed using the snap-cap. Place the plates or tubes in a moisturebarrier bag along with a silica gel desiccant packet. Samples properly sealed can be stored at ambient temperature with relative humidity below 40%.

Storage of DNA from Glycerol Stocks

Glycerol stocks containing E. coli strains can be transferred from cold storage into CloneStable for short term room temperature storage. The glycerol must be removed prior to storing the bacterial cells in CloneStable, as residual glycerol will interfere with complete drying. Due to interference from the residual glycerol stocks, better stability is obtained by growing glycerol stock cell in an overnight culture containing appropriate media and selective antibiotic. After testing the culture for appropriate plasmid content it can then be spotted into CloneStable for long term storage.

  1. Thaw glycerol stock and separate cells from the glycerol solution by centrifugation at 6,000 x g for 2 min. A cell pellet is formed with clear solution above it. If the solution appears cloudy, spin the sample for a longer period of time.
    Note – stocks with high concentrations of glycerol (>50%) must be diluted with water so that the glycerol content is <50% before pelletting the cells. Cells will not pellet if the glycerol solution is too viscous. Remove the supernatant.
  2. Resuspend pellet in desired volume of water. We recommend resuspending the pellet to an OD600 = 2.0 (~2x109 cells /ml) and spotting a 20 µl volume onto CloneStable. This gives a final cell density of ~4x107 cells per well.
  3. Heat Lysis (optional). Bacteria are viable in CloneStable in a dried state for up to a month. If the sample will be used for transformation into a new strain within the first month of storage heat the bacterial suspension at 80°C for 5 minutes prior to application to ConeStable to completely lyse the cells.
  4. Add resuspended pellet to CloneStable. Allow the matrix to be hydrated by the aqueous sample for 5-10 minutes. We recommend spotting a 20 µl volume into CloneStable.
  5. Mix the well or tube by gentle pipetting, and avoid forming bubbles.
  6. Dry the uncovered sample completely at room temperature. We recommend using a laminar flow hood (see table 1 above for drying times) or drying with a vacuum concentrator (see 2able 2 above for drying times).
  7. Cover samples after drying and store at room temperature. After complete sample drying, plates should be re-sealed with aluminum foil seals (provided in kit) to protect the sample. Tubes can be closed using the snap-cap. Place the plates or tubes in a moisturebarrier bag along with a silica gel desiccant packet. Samples properly sealed can be stored at ambient temperature with relative humidity below 40%.

Recovery of Bacterial DNA

  1. Add 20 µl of water or aqueous buffer directly to the dried sample in CloneStable. Alternatively, samples may be rehydrated directly with aqueous buffers used for downstream applications.
  2. Incubate at room temperature for 15 minutes to allow complete rehydration. After 15 minutes, mix the sample by gently pipetting up and down. The rehydrated sample is now ready for use directly in downstream applications.
  3. Store unused rehydrated samples at 4°C or room temperature for up to 10 days. Rehydrated samples contain CloneStable and can be re-dried without loss of efficient sample stabilization. We do not recommend repeating the rehydration/drying process more than 3 times per sample. Samples can also be stored at –20°C for long term storage.

Materials

     
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