I have a DNA origami structure which i need to validate its proper folding. I am having a tough time visualizing the structures using TEM. I suspect the reason being bad sample prep.
Hello Samer
i tried it once but couldnt visualize anything. Can you be explicit with the protocol like how to do it?
thanks and regards
Hi Prakash,
What sample preparation protocol are you using now?
Have you done any other quality control on your sample before TEM ? I would recommend you to first do Agarose gel electrophoresis if possible to assay your folding.
Also in my experience removing the excess staples after folding will greatly improve your TEM image quality, this can be done by gel extraction or spin filtration.
Hope this helps you!
Erik
Dear samer,
i guess my incubation timing for the negative staining was really low. this will definitely help. thanks and cheers
Dear Erik
i did run them on the gel and they look promising. and i am using a glycerol density gradient centrifugation method to get rid of the excess staples. it works fine for me. Do you think the residual glycerol in the sample would interrfere in the TEM imaging?
Hi,
Yes, if you use a sample from a glycerol gradient directly for TEM, I suspect the glycerol can affect your TEM samples. When I have used glycerol gradient centrifugation I have removed the glycerol by dialysis or spin filtration before TEM imaging. Glycerol gradient centrifugation may not be the most simple way to remove staples from an origami sample, for simple TEM studies agarose gel extraction should be enough.
Have you tried TEM on the sample without removing the staples? This is should be no problems.
As for the incubation times in the TEM sample prepartaion, we normally only leave our samples on the grid for 20 seconds and only stain in uranyl formate for 20s.
It may be helpful if you share the protocol you are using now so we can comment on it.
Erik
Hello Erik.
thanks for the inputs. I am also following the same thing as you did. take ~5uL of the sample containing folded structure(10nM) and put them on grid for 10 minutes. Wash off the excess samples with paper and apply uranyl acetate for 20- 30 seconds, let them dry overnight and image. while imaging i saw clumps presumably of salts.
thanks
Ok. At those concentrations of origami I usually only apply the sample for 20 seconds.
Was this done with a sample containing glycerol?
Did you filter your Uranyl Acetate solution?
TEM sample preparation can be a bit uneven, and parts of your grid may be useless while another part of the grid is fine.
Erik
well, i did not filter the stain which i am going to do the next time i prepare the samples. also i wanted to know if the integrity of the origami is fine with Agarose gel purification. and if any special TEM grids would help.
Thanks again for your time
Lama
Yes, the structures survive gel extraction with freeze and squeeze methods such as this:
http://www.bio-rad.com/en-us/product/freeze-n-squeeze-dna-gel-extraction-spin-columns
We use Formvar and carbon coated grids from electron microscopy sciences that we glow discharge for ~ 20 seconds before sample application.
Dear erik... Can you suggest me the best way to store the folded products for a long time.
thanks
Hi,
If your structures are not functionalized they should be fine in room temp for a few weeks.
I have also sometimes frozen samples for longer storage and this has worked fine for me but this is only limited experience.
Cheers
Erik
thanks erik
i unintentionally stored the stuctures with glycerol (purified from the density gradient) at 4 degrees for a couple of months and they looked fine as compared to the ones without glycerol.
Lama
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