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Hi,
I'm new to AFM and will be starting a research project visualizing protein–DNA interactions. I've been comparing different methods of preparing DNA and a freshly cleaved mica surface for AFM imaging (in solution using tapping mode), and it seems the most commonly used procedure involves treating the mica with NiCl2 and preparing the DNA in a MgCl2 buffer. Does anyone have a preferred protocol or methods paper that I could use to help get started? I've checked out the articles on the Veeco website already, and any other tips would be greatly appreciated. Thank you! Morgan
Hi Morgan~
Yes, the most common method of immobilizing DNA on mica is to use divalent cations, like NiCl2 and MgCl2, to passivate the negative charge on the mica and thus allow adsorption of the negatively charged DNA. Other groups use what we call "AP-mica" which is mica that has been silanized with an amino-terminated silane to give the mica a positive charge. I have uploaded 2 documents that have detailed procedures for preparing both types of DNA samples.
DNA Sample Preparation_102809.pdf
AP-MICA Preparation.pdf
Good luck with your experiments!
Andrea
Hi Morgan,
As Andrea said, you can use either the divalent cations or the APTES-modification methods. Regarding what you originally said, I think kit's best to use EITHER Nickel OR Magnesium, not both, if that's what you meant. I do not know if using both will cause some competition problems, but it is simpler to stick with one or the other. Like Andrea's method, I tend to find Nickel more effective than Magnesium. Note that there is a section on sample prep. for AFM, including a bit about sticking down oligonucletodes and proteins, including the original references to both these two techniques in my book, which you can read about here: OUP.com.
Good luck!
Pete.
Thank you for your help! I'll try using the Nickel.
Thanks again,
Morgan
These were very helpful. Thank you. I'll let you know what ends up working best.
Thanks,
Hi again,
We are going to be using Silicon Nitride probe tips to image DNA in a buffered solution. I was wondering which tip you have found works best for this?
Morgan,
The SNL levers work great. The c-lever is the one you want to use for TappingMode operation. http://www.veecoprobes.com/p-3693-snl-10.aspx
Thank you very much. This is very helpful!
Hi Andrea,
I am an undergraduate doing research on DNA imaging using an AFM. I would like to ask your permission to use your DNA sample preparation procedure. Also, I am wondering the type of probe used?
Thank you
Jenna