Hi
I'm not having any success (Nothing visible on SEM) binding amine bound 1.4nm nanogold to DNA origami structures. I'm currently using a 100Kd protein concentration column, where origami and gold are loaded together, and a series of spin washes to bind the structures and purify the sample which can then be recovered from the columns collection chamber.
If I go down the road of applying structures directly to the silicon slide does anyone have any advice in regards to fixing and washing the slide? I have a gold enhancement kit which will require washing the sample in H20 once its been applied.
At this point any advice or thoughts would be more than welcome.
Thanks for your time
Dear Simon Butler,
Although it is feasible, observing DNA on SEM is a little bit challenging. Try cleansing your silicon substrate with RCA method (just the basic part should be enough). It will make it more hydrophilic (never touch it, not even with gloves - use tweezers always).
You can try adding some microliters of your purification product directly onto silicon and let it dry (or rest for some minutes - try both). Then use your gold enhancement kit. Be gentle, otherwise the DNA will detach.
Be sure you are using a FE-SEM in order to get the best results. Use low kV and low current to avoid DNA damaging.
An easier alternative is using AFM (atomic force microscopy) if you have access.
Please let us know your progress. Good luck
Hi Wagner Correr
Thanks very much. Thats very helpful.
If you could spare a few more moments:
When you say the basic part of the RCA method do you mean just the initial particle clean?
Would you say that once DNA has been applied it is then possible to wash the slide with water in an enhancement step or would this just wash away the DNA?
Thanks again
Simon
Yes. I meant the first part: 5 parts of deionised water, 1 part of H2O2 , and 1 part of NH4OH. Place the silicon pieces inside and bring it to 70-80 ºC during 10 minutes. Rinse with deionised water.
To wash the slide, you can carefully submerge it in water or, using a micropipete, put a drop on it and take it back gently. If there is too much turbulence you will detach the DNA.
Thanks very much for your help in this matter Wagner Correr. Since this discussion I successfully visualised ~100 nanometer structures created from an array of helices. Unfortunately I was naive in assuming aggregation of gold would be less on the tailing single strands of the DNA origami scaffolds and the structures are very messy. However having confirmed the design I am now going to set about utilising the remaining scaffold into a larger lattice reinforcing the design and using thiol/oligo bound gold nanoparticles to attach in a site specific manner.
I will also hopefully be conducting AFM of the design at some point which will add additional characterisation of the project.
Again Thanks.
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