1Laboratory of Molecular Genetics, CNRS UMR 8541, Biology Department, Ecole Normale Supérieure, Paris, France, 2DSV-IBitec-SBIGM-LMARG, Commissariat à l’energie atomique (CEA), Saclay, France, 3Physique de la Matière Condensée, École Polytechnique, CNRS, Palaiseau, France, 4Institut de Recherche Interdisciplinaire, Cité Scientifique, Villeneuve d’Ascq Cedex, France
H.L.’s present address is Marie Curie Research Institute, The Chart, Oxted Surrey RH80TL, UK
Peer-reviewed,author-submitted protocol Published in November 2009 (p.48) DOI: 10.2144/000113255
Removal of contaminants on coverslips
1. Loosely place 25 coverslips (22 × 22 mm) into a clean 250 mL beaker.
2. Rinse the coverslips briefly in acetone and transfer them into a new beaker.
3. Add 50 mL 50% methanol/50% water and sonicate in an ultrasonic bath for 20 min.
4. Transfer coverslips to a new beaker and sonicate for 20 min in 50 mL chloroform.
5. Dry coverslips on chloroform-cleaned glass plate.
Cleaning the coverslips in piranha solution
6. Place dry coverslips loosely, without a special rack, in a chloroform-cleaned, dry glass beaker and fix it on a support stand via clamp holder and utility clamp, as illustrated in Figure 1. Place the coverslip-holding beaker in a much bigger beaker containing warm water (~65°C).
7. Piranha solution (50 mL) is made of 70% sulfuric acid (99% w/v) and 30% hydrogen peroxide (35% w/v). In a fume hood, first add the peroxide solution into the beaker containing the coverslips. Then, slowly pour the acid into the beaker. Close fume hood. The reaction is exothermic and small bubbles immediately appear, indicating the start of the oxidation reaction. Mixing and further heating is not required. Leave coverslips in the reactive solution for 20 min.
ATTENTION: Most organic products are hazardous and need to be handled with care under a working fume hood. Special safety measures should be taken during the preparation of the piranha solution. Piranha solution is explosive when in contact with organic reagents. The fume hood should be emptied of all unnecessary equipment and reagents, cleaned with chloroform and dried. All beakers and tweezers in contact with piranha solution should also be cleaned with chloroform.
Preparation of the piranha solution is also performed under a fume hood. Pouring the sulfuric acid should not be done too quickly, but not too slowly either. As its name suggests, this is a very aggressive, corrosive mixture that is potentially unstable if used carelessly. Therefore, the operator should wear appropriate laboratory protections (lab coat, latex gloves, and safety glasses).
8. Recover the coverslips from the beaker with Teflon-coated tweezers (one should avoid tweezers made of other polymers or nickel as piranha solution reacts with them) and sonicate for 10 min in chloroform in a new clean beaker.
9. Transfer coverslips one by one in a new beaker with ultrapure water and sonicate for 5 min.
10. Transfer coverslips one by one in a new beaker with chloroform and sonicate for 5 min.
11. Transfer coverslips again to water one by one and sonicate for 5 min.
12. Transfer coverslips one by one to chloroform and sonicate for 5 min.
HINT: Transfering coverslips one by one in new solutions from Step 9 onwards is essential to remove viscous acid. Make sure that no viscous acid residue is left on the coverslips after Step 12. Otherwise, repeat Step 11 and Step 12.
13. Thoroughly dry coverslips on a chloroform-cleaned, dry glass plate.
14. For disposal, the piranha solution is slowly diluted in 3–4 L water and neutralized with 30% NaOH.
Alternative procedure for cleaning with piranha (for steps 6–14): cleaning of the coverslips in a plasma cleaner
Alternative Step 6: Place coverslips one by one on glass plate and expose it to plasma under vacuum for 1 min.
Alternative Step 7: Turn coverslips and expose to plasma again for 1 min.
Alternative Step 8: Place coverslips in a large beaker one by one and dehydrate at >100°C for at least 1 h in an oven. Proceed to step 15.
15. Place rapidly dried coverslips in clean beaker.
HINT: In order to avoid rehydration of coverslips, which affects proper silanization, proceed quickly from Step 14 to Step 15, or Alternative Step 8 to Step 15.
16. Add 100 mL n-heptane and 100 µl of (7-octen-1-yl)trimethoxysilane to beaker.
HINT: The beaker containing the silanization solution should not be sealed with parafilm or aluminum foil, as vapors of heptane and the silane solution, respectively, dissolve the parafilm and corrode the aluminum foil.
Once opened, silane vials are stored in a desiccator without vacuum for no more than 2 months (after 1 month, we have sometimes observed a decrease in the quality of DNA fiber alignment). Alternatively, silane can be stored in argon or nitrogen in order to avoid oxidation and polymerization.
17. Place beaker overnight in a desiccator, without vacuum, under a chemical hood.
18. Transfer coverslips one by one into a new beaker with n-heptane and sonicate for 5 min.
19. Transfer coverslips one by one into a new beaker with distilled water and sonicate for another 5 min.
20. Transfer coverslips one by one into a new beaker with chloroform and sonicate for 5 min.
21. Dry coverslips and keep them in a carefully closed container. The surfaces should be free of any white residue; if they are not, the coverslips should be discarded.
HINT: Coverslips can be successfully used for molecular combing of DNA for 2–3 months after silanization when stored in a dark, dry place at room temperature.
Most problems with DNA combing arise from the quality of silanized coverslips. Batches should be tested and the optimal pH of the MES solution should be calibrated using standard DNA (i.e., Lambda DNA stained with YOYO-1). However, optimal pH varies with different types of DNA, from pH 5.9 to 6.3.
To simply test the hydrophobicity of coverslips, deposit a water drop on a silanized coverslip. The drop should adopt a characteristic round form, distinct from the flat drops that form on untreated glass.
1. White residues on coverslips after step 20: start again and do more extensive washing after the piranha step.
2. Coverslips are not very hydrophobic: start again with fresh silane stock, not opened stock.
• Glass coverslips (Erie Scientific, Portsmouth, NH, USA)
• Fresh Milli-Q ultrapure water
• Methanol, CHROMASOLV Plus for HPLC, ≥99.9% (Catalog no. 646377; Sigma Aldrich France, Saint Quentin Fallavier, France)
• Concentrated sulphuric acid, 99.999% purity (Catalog no. 339741; Sigma Aldrich France)
• Hydrogene peroxide solution, 35% wt. in H2O (Catalog no. 34988-7; Sigma Aldrich France)
• Chloroform, CHROMASOLV Plus for HPLC, ≥99.9% (Catalog no. 650498; Sigma Aldrich France)
• Anhydrous heptane 99% purity (Catalog no. 246654; Sigma-Aldrich France)
• (7-octen-1-yl)trimethoxysilane (Catalog no. 452815; Sigma Aldrich France)
• Acetone (Catalog no. 20066.296; VWR France, Fontenay-sur-Bios, France)
• Glassware (Fisher Scientific, Illkirch, France)
• Support stand, clamp holder, utility clamp (Fisher Scientific, Illkirch, France)
• Oven (Roucaire, Courtabouef, France)
• Glass desiccator (VWR, France)
• Teflon tweezers (Catalog no. 232–0022, VWR France)
• Ultrasonic bath (FS6, Catalog no.142–6044, Fisher Scientific)
• Plasmacleaner PCD-002 (Harrick Plasma, Ithaca, NY, USA).
Address correspondence to Kathrin Marheineke, Laboratory of Molecular Genetics, CNRS UMR 8541, Biology Department, Ecole Normale Supérieure, 46 rue d’Ulm, 75005 Paris, France. email: [email protected]
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