AL's+Week+of+March+3

__3/3/08__ - So far I've been working on my Science Fair such as my research plan and abstract as well as analyzing images. Today I made graphs for my analysis, which was both the average plasmid distribution on my sample surfaces and the avarage plasmid length. I included eror bars that I calculated on my graphing calculator.

__3/4/08__ - I will post images of the graphs once I figure out how to upload the images without them turning completely black.

In the mean time, I will post my research plan.


 * //2007-2008 Research Plan//**

//The problem of my project is that a positively charged solution called APTES (Aminopropyltriethoxysilane, C////9////H////23////NO////3////Si) is used to bind DNA plasmids (circular DNA which has a negatively charged phosphate backbone) to a silicon surface (also negatively charged), but it binds too tightly, making the DNA coiled and clumped thus difficult to analyze under the Atomic Force Microscope. How can the DNA plasmids be less tightly bound to the silicon substrate? The hypothesis is that other monolayer substances could be introduced in order to offset the APTES solution’s strong positive charge. These other substances are PEG (Polyethylene glycol) and OTS (octadecyltrichlorosilane). The other aspect of my project is another strategy of getting the same results. This is to use restriction enzymes to linearize the DNA plasmid and use other deposition strategies when depositing the linearized DNA// //Depositing DNA on silicon is not as simple as it sounds, because any trace of dirt or even stray molecules will ruin the AFM image. In order to be adept at using the AFM to image DNA, one must follow the procedure thoroughly. The first step is to cut 4” silicon wafers into 1 cm.x1 cm squares, so they will fit on the AFM stage. Then scotch tape is applied to both sides of the silicon squares and then taken off to remove debris. The chips are boiled 30 min in toluene in a clean beaker with a watch glass on top, under the fume hood. This part removes the grease from the silicon wafers. The wafers should be rinsed with 18 Mega ohm resistance water after the wafers have cooled, then dry it off with N2 gas. Afterwards, 20 ml of concentrated H2SO4 will be added to six to seven ml of (30%) H2O2 (piranha acid) in a 50 ml beaker. This will destroy any organic molecules and build up an oxide layer. The dry wafers will be boiled in the piranha acid for 15 to 30 minutes at 90° C. I then cover the beaker with a lid. The wafers are then removed using plastic or metal tweezers and rinsed thoroughly with distilled H2O. I then dry them with N2 gas and dispose of the piranha acid into the acid waste bottle.// //Now comes another major part in the procedure. The wafers are moved to the MOS bench in the clean room where the RCA1 and RCA2 baths should be turned on. The RCA1 and RCA2 baths are located on a counter with two sinks on it. The wafers are first placed into the RCA 1 bath and the bubbles turned on for ten minutes. I rinse the wafers and tweezers for two cycles in distilled water. The wafers and tweezers are placed into RCA 2 (HCL and H2O) and bathed for ten minutes, and the bubbles are turned on again. Afterwards, the silicon wafers are rinsed for two cycles in H2O. What this process does is removes the natural uneven SiO2 layer and grows a new one nm thick layer on the wafers. When this is complete, the wafers are dried with N2 gas and they are stored in a clean container. Finally, I turn off the bubblers and heaters, clean up the bench for any spilled chemicals, and take off protective gear. This process was done under direct supervision by a superior.// //Growing a monolayer of APTES was the main focus of this project last year. Now the APTES monolayer is mainly used to compare to the new monolayers I am using, which are the PEG (Polyethylene glycol) and OTS (octadecyltrichlorosilane) monolayer. In making APTES, the first thing to do is to make a 1% solution of APTES in water. This solution has to be made fresh right before it is used. I first take 1980 µL of H2O and mix it with 20 µL of APTES into a clean vial, and place the wafers within the vial, and leave it there for around ten minutes. When the ten minutes are finished, the silicon wafers are washed off yet again with 18 Mohm H2O and dry with N2 gas. //

//The next part of the procedure is to add the DNA plasmid solution along with the buffer solution. 2 µL of DNA plasmid and 18 µL of buffer solution are added using a pipette into a vial and shaken. The buffer solution holds the biomolecules together in the case of silicon, however in the case of mica; the buffer solution is used as an adhesive because of the MG 2+ it contains. However, silicon is preferred to mica because it is necessary in creating DNA structures because of the current it conducts. The DNA and buffer mixture are left on the silicon substrate for five to ten minutes. After, the substrate is showered with 18 Mohm H2O and then driedd again with N2 gas.// //Making OTS (octadecyltrichlorosilane) solution is somewhat more complex, as it uses an oxygen free “dry box.” The first step is to acquire some vials to be used to contain the silicon wafers. Depending on how many samples are to be analyzed, the number of vials used will differ. It is best the vials are labeled appropriately so that it is known which samples are which. Before doing anything with them, they are cleaned using about 20 mL of piranha acid and are rinsed out with 18 ohm water and put in an oven after being wrapped with tin foil overnight. This is to get rid of any excess moisture that might contaminate the dry box. Afterwards, I go to the dry box where there is a small vacuum chamber attached to the large box itself. Before doing anything, the vacuum chamber should be refilled with nitrogen gas by pulling a lever. The already cleaned silicon wafers are put in the cleaned vials, the vials closed, and then put into the vacuum chamber attached to the dry box. This process is to vacuum out any potential moisture. To do this, a lever is pulled towards the “evacuate” mode and the samples is left to sit for around 8 minutes. The lever is turned to make the dial go to “-25”, then slowly turned again to evacuate (second pump down). I then wait for about 4 minutes. Finally, the knob is turned slowly until the dial says “-7” and the samples are taken out from the other end by putting my hands into the gloves attached to the dry box and by turning the large knob from the inside of the dry box. Inside the dry box itself there should be some OTS solution in a bottle. 5 mL of this solution should be dispensed into each vial with the silicon wafer still inside the vial. The samples are left there for a few days, and when it is taken out, the wafers are cleaned, and then dried. I normally deposit DNA on the OTS monolayer and take images of the samples on the AFM.// //The other monolayer I deposited on the silicon wafer was PEG solution, which I essentially did two ways. The first way, I deposited the PEG solution with water by mixing 20 μL of the PEG solution with 1980 μL of water. As a variable, I submerged the silicon wafers in this PEG solution for varying amounts of time, and deposited DNA to see how it reacted to the different deposition times. In the other way, I deposited 2 mL of Toluene, 40 μL of PEG and 10 μL of HCL to act as a catalyst into a vial. I then submerged the silicon wafers into the vial and left it there for one to three days, depending on the sample.// //Minor tools used in this project are the ellipsometer which measures the height of the monolayer, and the contact angle machine, which says whether or not there is a monolayer by measuring whether the surface is hydrophilic or hydrophobic. The ellipsometer is a large instrument with a number and letter dial on the upper right side with a screen that says what characters one has put in. In order to properly use the instrument, the bare silicon surface of the sample with no monolayer must be measured first. The silicon wafers are put on the large platform and the laser is focused on the sample using three wheels on the sides of the platform. Press the program button first, followed by 2-1-1, then hit the button “E” (enter). When the screen asks, “N?” 1.46 is typed and “E” is hit again. I then usually pick another point with the laser and press the button “continue” so that it will take another measurement. Afterwards, the sample with a monolayer (APTES, OTS, or PEG) is put in and focused on with the laser once again. On the dial, the buttons “program”, 2-2-1, and then “E” are pressed in that order. When the screen asks for “upper N?” 1.5 and then “E” should be pressed again. The ellipsometer will then ask, “low film T?” The measurement that is previously acquired from the bare silicon sample should be used. Finally, the ellipsometer will ask, “lower film N?” Here, the number 1.46 should be pressed, and then the ellipsometer will take the height measurement of the monolayer in angstroms.// //The contact angle machine is normally used to make sure the monolayer on your sample has grown across your sample. Certain substances are known as hydrophobic and hydrophilic. Hydrophilic monolayers attract a lot of water, causing water droplets to spread all over the surface, and hydrophobic monolayers will cause water droplets on the surface to become tense and bead up. The contact angle machine measures the angle relative to the surface of water droplets that are put on the sample so you will know whether the surface is hydrophobic or hydrophilic. Angle measurements greater than 90 degrees are hydrophobic and angles lower than 90 degrees are hydrophobic. For example, we know that APTES is hydrophilic and that the measurements should be somewhere around 60 degrees, so if you get these measurements, you know you have an APTES layer on your sample. To use the instrument, the sample is put under the pipette on the platform. The needle is filled with water, and another knob is used to bring the needle in, the needle is depress using yet another knob in order to bring it close to the sample, and a droplet of water is put on the sample. The microscope attached to the instrument where there is a protractor is peered through. The sample should be focused on and the protractor crosshairs are used by turning a black knob to measure the angle itself and write it down. About four measurements should be taken all together for each sample and averaged.// //** The second part of my project besides using and analyzing different monolayers, was to use restriction enzymes to cut the DNA plasmid at one point in order to linearize the plasmid. By doing this, I hope that the effects and changes that the monolayer does to the DNA will be more visible due to the fact that the DNA itself will appear longer under the AFM. Also, the linearized DNA should be freer to relax than the tight, circular, plasmid. In order to use restriction enzymes on the DNA one must follow a certain procedure. First, 5 **μL of the** Kpn I restriction enzyme, 10 **μL of buffer solution, 1 μL of bovine serum albumin (BSA), 5 μL of 1 μg/μL concentration DNA plasmid, and 79 μL of water are taken and put into a tube. The kpn I enzyme is used because it cuts the DNA plasmid in only one spot, and the enzyme is readily available. The BSA is used as it adds extra proteins to stabilize the enzyme. First, the tube is put in 37° C temperature distilled water for 12 hours, then taken out and put in freezer. 37° is the optimum incubation temperature for the enzyme. The tube is then placed in ice.100 μL of phenol (ph level 8) should be taken and mixed into this the solution for 5 minutes. The phenol will stop the reaction and gets rid of the protein. The DNA is then put into the centrifuge and put in a tube of water in the opposite end for about 5 minutes as this brings the dissolved particles to the bottom of the tube. Then, 100 μL of this solution is taken out and put it into another tube and another 100 μL of chloroform isoamyl alcohol added and put into the tube and the tube placed in the vortex again. The tube is the inserted into the centrifuge again for only 1 minute. The top 100 μL layer (you should see different concentrations in the tube/layers) is taken and the substance is put into yet another tube. 3 M of sodium acetate (CH3OO- Na+) are added. 3 M is equivalent to 10 μL. 300 μL of Ethyl alcohol or ethanol are then added to that. Afterwards, put it in the -20° C freezer for either for 1 hour or overnight. After being put in the freezer, the tube is taken out and put in the centrifuge in the cool room for 20 minutes. The ethanol is removed from tube, and there should be a pellet in at the bottom. The pellet is washed in cold 70% ethanol and the pellet is washed in 20 μL of water. Then 5 μg of DNA are put in the 20 μL of water. This DNA should normally be deposited the same way one deposits plasmid DNA on silicon. As a part of my project, I deposited the linearized DNA in different ways, such as putting a cover slip on the droplet of DNA, or using nitrogen gas to blow on the substance, both when on the silicon. I did this in the hopes of getting the DNA to straighten out rather than coil.// //The final stage in the process is looking at the DNA under the AFM and analyzing the sample. The first step is to take a small cantilever and slide it into a small cantilever holder using small clean tweezers. The cantilever contains a tip that analyzes the sample in the AFM. The cantilever holder should then be inserted into the AFM. The sample is taped to a magnetic puck and placed directly under the tip holder also using the tweezers. There is a small camera next to the AFM that shows a picture of what the camera and tip see and what part of the sample the AFM is focused in on. There is a red laser that appears in the AFM that must be directed on top of the tip so the AFM can process the information analyzed by the cantilever tip. On the base of the AFM there is a dial and by turning the knobs on the dial, the number will change. The number should read as close to zero as possible in order for the information to be processed correctly and shown on the computer. the Nanoscope software should be brought up on the computer screen. The next step is to tune the tip, which can be done by simply pressing a button that is labeled appropriately. I then go to the screen and have the AFM bring the tip down to the sample by clicking the button “Approach sample.” The tip on the camera will move back and forth. Whatever debris or DNA the tip goes over will appear on the computer screen as different colors which denote different heights in the sample. By looking at the height and looking at cross-sections and different maps of the sample through the AFM software, it is easier to tell if the substances used have done their job. If the sample is cleaned well, it will be relatively smooth with little color variation besides the DNA samples which are about 2 nm in height. If a worthy or insightful image is found, a picture should be taken of it using the program feature that shows a button with a camera on it. When the image is fully analyzed by the tip, the camera button is pressed and then the picture will automatically be taken and saved to the disk. Afterwards, based on the data, the DNA deposition process is changed to hopefully correct problems that were seen with the DNA such as the plasmid or linearized DNA being too coiled.//