GENE DELIVERY
DNAdelTM
siRNAdelTM
CHLAMYDOMONAS
PROTOCOLS
REFERENCES
 
DNAdelTM Protocol | siRNAdelTM Protocol | Chlamydomonas Protocol

DNAdelTM Gold Carrier Particles

Seashell Technology Plasmid DNA Binding Particle Formulation Protocol S550d, S1000d, S1600d Gold

  1. The DNAdelTM gold particles are supplied as a 50 mg/ml suspension in binding buffer. Sonicate briefly to dissociate any aggregates prior to formulation.

    Dilute the gold into binding buffer to yield a final concentration of 30 mg/ml (minimum volume of 50 μl buffer; 1.5 mg gold (30 μl volume of 50 mg/ml; 20 μl buffer)). Add the plasmid DNA (stock 1mg/ml) to gold at a ratio of 2-5 μg DNA per mg gold. (This concentration has yielded significantly higher expression levels compared to standard particles and protocols). Vortex briefly.

  2. Add an equal volume of precipitation buffer. Vortex briefly and let stand for 3 minutes. Spin (10,000 rpm in Eppendorf microfuge 10 sec) to pellet the DNA coated particles.

  3. Remove the supernatant and add 500 μl of 100% cold ethanol. Vortex briefly and spin (10,000 rpm in Eppendorf microfuge 10 sec) to pellet particles. Remove the supernatant and add 100% ethanol to the desired gold concentration. Briefly sonicate the solution to resuspend the gold particles and process for appropriate delivery device The sonication step minimizes aggregation and is important for reproducible particle delivery.

Key Parameters for High Efficiency Transformation

Brief sonication prior to placement of the particles in the delivery device improves particle dispersion during delivery.

DNA quality is a critical factor for transformation efficiency; therefore plasmid preparations should be free from contaminating RNA and protein.

Store particles and buffers at 4°C. Sonicate and vortex the carrier particles prior to formulation.

DNAdel is a registered trademark of Seashell Technology, LLC.

This product is for research use only and is not intended for diagnostic use or to treat, cure or prevent any disease. The product is not available for resale. The safety of this material has not been determined and we recommend that this product and components are handled only by persons trained in laboratory techniques and are used in accordance with good laboratory practice. As all chemicals should be considered as potentially hazardous it is advisable when handling chemical reagents to wear suitable protective clothing. The BiolisticTM process is covered by patents owned by Dupont, Inc. and purchase of these products does not convey a license under these patents.





siRNAdelTM Gold Carrier Particles

Seashell Technology siRNA Binding Particle Formulation Protocol S550ri, S1000ri, S1600ri Gold

  1. The siRNAdelTM gold particles are suspended in binding buffer at a concentration of 50 mg/ml. Sonicate briefly to dissociate any aggregates prior to formulation. Resuspend the desired amount of gold carrier particles in binding buffer to yield a final concentration of 30 mg/ml. For example, to prepare 2 mg of particles, add 40 μl gold carriers to 26 μl of binding buffer.

  2. Add the siRNA to the gold suspension. The recommended ratio for particle saturation is: 200 pmol/mg S550ri, 100 pmol/mg S1000ri, 40 pmol/mg S1600ri.

    These values can be converted to microgram units. Assuming an average molecular weight of 686 per base pair for dsRNA, the molecular weight of a 21 mer dsRNA would be 14406. Therefore,

         1 mol of a 21 mer dsRNA= 14406 grams
         1 pmol of a 21 mer dsRNA= 0.014 micrograms
         200 pmol of a 21 mer dsRNA= 2.8 micrograms

  3. Vortex briefly and let stand for 1-2 minutes at room temperature.

  4. Spin (10,000 rpm in Eppendorf microfuge 10 sec) to pellet particles.

  5. Remove the supernatant and wash the gold pellet with 500 μl of ice-cold 100% 2-propanol. There is no need to resuspend the pellet at this step. Spin briefly in the microfuge, remove the supernatant and add 100% ethanol to yield desired gold concentration for loading in the respective delivery device.

  6. Resuspend the pellet with a brief (1-2 sec) burst using a water bath or probe tip sonicator.

  7. Load the carrier for ballistic delivery as instructed in the user manual and shoot particles into the desired target.

siRNAdel is a registered trademark of Seashell Technology, LLC.

This product is for research use only and is not intended for diagnostic use or to treat, cure or prevent any disease. The product is not available for resale. The safety of this material has not been determined and we recommend that this product and components are handled only by persons trained in laboratory techniques and are used in accordance with good laboratory practice. As all chemicals should be considered as potentially hazardous it is advisable when handling chemical reagents to wear suitable protective clothing. The BiolisticTM process is covered by patents owned by Dupont, Inc. and purchase of these products does not convey a license under these patents.





Seashell Technology S550d gold DNA formulation protocol
Optimized for Chlamydomonas transfection
(5 transfections)

  1. Add plasmid DNA (from a stock of 1 mg/ml in TE or water) to 50 μl of binding buffer. Saturation can be achieved with a ratio of 4 μg selection plasmid and 10 μg of reporter plasmid per 3 mg of gold carrier.

    It is critical that the gold carrier is added to the pre-mixed plasmid DNA to insure that both plasmids bind to the carrier particles.

  2. Add the S550d gold carrier to the DNA in binding buffer. The stock is at 50 mg/ml, therefore add 60 μl (3mg) of S550d carrier.

  3. Let stand for 1 minute on ice and then add 100 μl of precipitation buffer. Let stand for 1 minute.

  4. Mildly vortex and spin (10,000 rpm in Eppendorf microfuge 10 sec) to pellet particles.

  5. Remove the supernatant and wash the gold pellet with 500 μl of cold 100% ethanol. There is no need to resuspend the pellet at this step. Spin briefly in the microfuge, remove the supernatant and add 50 μl of ice-cold ethanol.

  6. Resuspend the pellet with a brief (1-2 sec) burst using a probe tip sonicator. Immediately transfer 10 μl to the carrier membrane. Keep aggregation to a minimum by sonication immediately before applying the gold to the carrier membrane.

  7. Place the macrocarrier in the first slot beneath the rupture membrane holder.

  8. Launch the particles using a 1350 psi rupture membrane and 9 cm distance from the macrocarrier.

This product is for research use only and is not intended for diagnostic use or to treat, cure or prevent any disease. The product is not available for resale. The safety of this material has not been determined and we recommend that this product and components are handled only by persons trained in laboratory techniques and are used in accordance with good laboratory practice. As all chemicals should be considered as potentially hazardous it is advisable when handling chemical reagents to wear suitable protective clothing. The BiolisticTM process is covered by patents owned by Dupont, Inc. and purchase of these products does not convey a license under these patents.





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