Dynatwist

• Dynatwist is provided as a 50 pM solution of scDNA molecules in Tris-HCl 10 mM pH 8.

• Dynatwist should be stored at - 80 °C until first use. After thawing for the first time, Dynatwist may be stored at 4 oC or dispensed into 5 – 10 μL aliquots and stored at - 80 °C. Performances are guaranteed for a total of 6 months from receipt.

• Dynatwist should be manipulated with the same care given to any other DNA-based reagents. In addition low binding tubes should always be used.

The 100 μL vial allows preparing around 200 samples similar to the one described below. It contains scDNA molecules ready-for-use for single-molecule experiments.

The present protocol is currently employed for micromanipulation with magnetic tweezers, Dynatwist 5 kbp (biotin, digoxigenin) or 17 kbp (biotin, digoxigenin) being tethered between the anti-digoxigenin coated glass surface of a flow chamber and streptavidin coated magnetic beads (e.g. Dynabead MyOne C1). It can easily be adapted to beads having a different size or made of a different material, depending on the targeted force range and on the used single-molecule setup.

1. Resuspend the 10 mg/mL stock of streptavidin coated magnetic beads by gentle pipetting.

2. Place in a small-volume low-binding tube 200 μL of Wash Buffer (20 mM K-Hepes pH 7.8, 100 mM KCl, 5 mM MgCl2, 0.5 mg/mL BSA, 0.1 % Tween-20, 2 mM DTT).

3. Add to the Wash Buffer 5 μL of resuspended magnetic beads and mix by gentle pipetting.

4. Use a rare earth magnet to pull the beads to one side of the tube and pipette away the supernatant.

5. Resuspend the washed beads in 35 μL of Wash Buffer.

6. Place 0.5 μL of the 50 pM Dynatwist solution at the bottom of a small-volume low-binding tube.

7. Using a cut micropipette tip, transfer the 35 μL of washed and resuspended beads onto the droplet of Dynatwist.

8. Gently invert the tube several times in your fingertips to allow gravity to disperse the beads to near-homogeneity.  

9. After an incubation time of 2 min, including the homogenization step, use the cut tip to transfer the bead and DNA mixture into the inlet reservoir of the flow chamber of your single-molecule setup.

10. After injection allow the beads to settle onto the anti-digoxigenin coated surface and then give them a few additional minutes. The sedimentation and the incubation steps typically take 5 min each.  

11. Wash out unbound beads by injecting Wash Buffer into the flow chamber, typically up to 2 mL in total are necessary.  

12. Inject the buffer you use for your experiments and apply some force. Your supercoilable double-stranded DNA molecules are ready for biophysical investigations.

Additional details and tips can be found here