Deposit+the+DNA

A major step in doing things for this project is depositing the DNA plasmid. Whether or not the DNA is linearized, this part of the process is the same. The DNA plasmid concentration used in this and every other process involving DNA plasmids is .01 µg/ µL. This means for every microliter (1 x 10−6 Liters) there is .01 micrograms of DNA plasmid. Both the plasmids and the buffer are liquids.

1.) The first step is to take the tube with the puc 19 DNA plasmid out of the freezer, and also take an empty tube to put the DNA plasmids in.

2.) The second step is to take a micropipette and extract 2 µL of the DNA plasmid and place it in the other tube.

3.) Next, open the vial with the buffer solution TAE/mg2+ and extract 18 µL of the buffer solution and place it in the same tube you just put the DNA plasmid in. You now have a total of 20 µL in this vial. For the plasmid to buffer, you should have a 1:9 ratio.

4.) Put the plasmid tube from the freezer back in the freezer and the buffer solution vial back in its place. Take the new vial that contains both the plasmid and the buffer, and press it on the vortex in order to mix up the solution. Do this for 5-10 seconds.

5.) There are a couple of options here.


 * a.)Take the micropipette and set the dial to (2+18) 20 µL to make sure that you get all of the solution, and put the solution on the surface of the silcion wafer, and let the DNA deposit on the surface for 10 to 15 minutes. This is the normal and most used way.**

b.)This should be done only with linearized DNA. Deposit the DNA ( 20 µl) on the surface, but place a coverslip on the droplet of DNA-Buffer solution, and drag it across the surface of the silicon chip 1 or 2 times. You can do this in the hopes of straightening the DNA plasmids out and this would allow the DNA to be better analyzed.

c.) This should also only be done with linearized DNA. After depositing the droplet of solution, continuously blow the droplet of solution on the wafer for 10 to 15 minutes, to see if the linearized DNA straightens out. This is simply another way to hopefully get the results wanted in step b., if that does not work.

6.) Afterwards, rinse the silicon wafer in 18 mega ohm water, then dry the wafer in Nitrogen gas to make sure that all the excess particles and DNA that did not adhere to the silicon chip are gone.

This is an image of linearized DNA on silicon, with the plasmid concentration of .025 µg/µL, more than the usual.This is why there is such an abundance of DNA on this 5 by 5 µm scan on the AFM. This sample went through the deposition process outlined in 5a. **I've circled the clearest examples** of the linearized DNA to give you a better idea of what the DNA looks like. Also notice how the DNA is not very relaxed and it is rather coiled in itself, making it rather difficult to make out the DNA plasmids.



Next Milestone - Explain Linearized DNA