Preparação de ADN de poliacrilamida reticulada-hidrogéis
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Summary
Abstract
Introduction
Protocol
Representative Results
Discussion
Disclosures
The authors have nothing to disclose.
Acknowledgements
Materials
| ssDNA | Integrated DNA Technologies (Coralville, Iowa)<br/>idtdna.com | Do not vortex ssDNA. Gentle invert the vial and/or pipette solution to mix. | |
| PBS with calcium and magnesium | Any brand. | ||
| 100x Tris-EDTA buffer (TE buffer) | Sigma-Aldrich (St. Loius, MO)<br/>sigmaldrich.com | T9285 | |
| 10x Tris-Borate-EDTA buffer (TBE buffer) | Sigma-Aldrich (St. Loius, MO) | 93290 | TBE is a reproductive toxin. |
| 40% Acrylamide solution | Fisher Scientific (Pittsburg, PA) | BP14021 | Acrylamide is a toxin. |
| Ammonium persulfate (APS) | Sigma-Aldrich (St. Loius, MO) | A3678 | Prepare a 2% solution in TE buffer. APS is a toxin and irratant. |
| Tetramethylethylenediamine (TEMED) | Sigma-Aldrich (St. Loius, MO) | T9281 | Prepare a 20% solution in TE buffer. TEMED is flammable, a corrosive, and a toxin. |
| 12 mm diameter round coverglass | Fisher Scientific (Pittsburg, PA)<br/>fishersci.com | 12-545-82 | |
| Norland optical adhesive 72 | Norland Products (Cranbury, NJ)<br/>norlandprod.com | NOA72 | |
| 24-well tissue culture plate | Any brand. | ||
| Microcentrifuge tubes | Any brand. | ||
| Sulfo-SANPAH | ProteoChem or Thermo Fisher, (Rockland, IL)<br/>proteochem.com or thermofisher.com | C111 or 22589 | Prepare a 0.315 mg/ml solution in water immediately before use. Dissolve at 37 °C and filter sterilze. It is normal to observe undisolved sulfo-SANPAH in the filter. Sulfo-SANPAH is light sensitive and, therefore, the solution should be protect from light until UV exposure. |
| Poly-D-Lysine (PDL) | Sigma-Aldrich (St. Loius, MO) | P6407 | Prepare a 0.2 mg/ml solution in water and filter sterilize. |
| Collagen Type I | Affymetrix (Santa Clara, CA)<br/>affymetrix.com | 13813 | Prepare a 0.2 mg/ml solution in 0.2 N acetic acid. Solution needs to remain cold at all times to avoid polymerization. Acetic acid is a flammable, toxic, and corrosive. |
| 22 x 60 cover glass | Fisher Scientific (Pittsburg, PA) | 12-544-G | |
| Positive-displacement pipette | Gilson, Inc (Middletown, WI)<br/>gilson.com | F148504 | |
| Heat block | Fisher Scientific (Pittsburg, PA) | 11-718 | |
| UV light source | Place gels as close as possible to the UV light. UV light can cause skin or eye injury. | ||
| Thermometer | Any brand. | ||
| Nitrogen gas | GTS-Welco (Flemington, NJ)<br/>www.praxairmidatlantic.com/ | NI 5.0UH-R |
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Cite This Article
Previtera, M. L., Langrana, N. A. Preparation of DNA-crosslinked Polyacrylamide Hydrogels. J. Vis. Exp. (90), e51323, doi:10.3791/51323 (2014).