Biomod/2013/Aarhus/Materials And Methods/System In Action

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System in action

Assembly of origami baseplate with cholesterol

2 µL of MgCl2 (125 mM) reaction buffer was mixed with 1 µL of M13mp18 (418 nM). 1.5 µL of BG1 staples (3.55 µM), 1.5 µL of BG2 staples (3.55 µM) and 1.5 µL of BG3 staples (3.55 µM) were added, followed by 1 µL of Dome staples (5 µM) and 1 µL of photosensitizer-ModLeft staples (5 µM). 6.5 µL of 0.5× TAE was added to a total volume of 20 µL. The reaction was subsequently incubated for 17 hours in a PCR machine with a non-linear time ramp and purified with 100 kDA Amicon filters.

Assembly of origami baseplate with photosensitizer

In order to assemble our origami baseplate with the photosensitizer-modified staple strands, 2 µL of MgCl2 (125 mM) reaction buffer were mixed together with 1 µL of M13mp18 (418 nM) (scaffold strand). 1.5 µL of BG1 staples (3.55 µM), 1.5 µL of BG2 staples (3.55 µM) and 1.5 µL of BG3 staples (3.55 µM) were added, followed by 1 µL of Dome staples (5 µM), 1 µL of PepLock staples (5 µM), 1 µL of 5 µM ModRight staples and 1 µL of photosensitizer-ModLeft staples (kan henvises til Mors påfund) (5 µM). 8.5 µL of 0.5× TAE were added to a total volume of 20 µL. The reaction was subsequently incubated for 17 hours in a PCR machine with a non-linear time ramp and purified with 100 kDA Amicon filters

Labeling of staple strands with terminal transferase

A master mix consisting of 26 µL 5× TdT reaction buffer (Roche), 26 µL CoCl2 (25 mM), 6.5 µL TdT Stock and milliQ H2O to a total volume of 130 µL was prepared. 1 mM Cy3-ddUTP or 1 mM Cy-5-ddUTP added to the mix. For the TdT-reactions with Cy-3-ddUTP, 13 µL staples of Modleft (10 µM) was added and in the TdT-reaction with Cy-5-ddUTP, 13 µL staples of ModRight/Purple (10 µM) were added. The reactions were incubated at 37˚C for 40 minutes and subsequently stopped by adding 13 µL EDTA (0.2 M).

Staining the samples for confocal microscopy

KB-EGFPluc-Wagner were seeded out as 8x104 cells/well in an eight well chamber slide. 24 hours after seeding out, the cells were transfected with the origami plate with attached cholesterols to a final concentration of 0.5 nM origamis baseplate for 2 hours. The medium was removed and the cells were washed twice with PBS buffer with 1 % sodium azide. The cells were then stained with 5 µg/mL Wheat Germ Agglutinin, Alexa Fluor 488 Conjugate (WGA-A488) (Life Technologies) for 10 minutes at 37˚C. The cells were washed twice in PBS buffer with 1% sodium azide. Fixation was done by adding 4% PFM and left for 15 minutes at room temperature to fixate. PBS buffer was used to wash the cellw once again. The well cage and gasket from the chamber slide were removed and dipped in a tube of ddH2O to remove salt. The slide was left to air dry for 20 minutes until the sample was approximately 90 % dry. 5µl/cm2 of Prolong Gold with DAPI (Life Technologies) were added on the slide. A cover glass was placed on the edge of one side of the slide. The slide were incubated until the next day in the dark room at room temperature. As the cells could not be visualized until some time later, the slide was treated with nail polish and stored at 4˚C.


References

  1. Jahn, K. et al. Functional patterning of DNA origami by parallel enzymatic modification. Bioconjugate Chem. 22, 819–823 (2011).[1] [Jahn]

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