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| __TOC__ | | __TOC__ |
- | '''Protocols'''
| + | <font face="Comic Sans MS"> |
- | ::Below, please find the steps that we followed to carry out molecular cloning, create recombinant DNA parts, and construct microfluidic channels.<br>
| + | |
- | | + | |
| =Molecular Cloning Protocols= | | =Molecular Cloning Protocols= |
| ==PCR Reaction== | | ==PCR Reaction== |
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| | | |
| =Microfluidics Protocols= | | =Microfluidics Protocols= |
- | <html>
| + | ''Protocols "Making an SU-8 Master of Your Design," "Making PDMS," and "Sealing Device" provided by Dr. Archer (Cornell University, Biomedical Engineering Department), from BME3010 lab class.'' |
- | <div>
| + | ==Making an SU-8 Master of Your Design== |
- | <ul style="margin-left:.0208in;direction:ltr;unicode-bidi:embed;margin-top:
| + | :'''Materials''' |
- | 0in;margin-bottom:0in">
| + | ::*A pretreated silicon wafer that has been cleaned with acetone and isopropyl alcohol (IPA) and dehydrated at 200oC for 10-20 minutes. |
- | <p style="margin:0in;font-family:"Comic Sans MS";font-size:10.0pt;color:black"><b><ul style="margin-left:.0326in;direction:ltr;unicode-bidi:embed;margin-top:
| + | ::*Hot plate – make sure it is set to 65oC (80C for VWR hot plates) |
- | 0in;margin-bottom:0in">
| + | ::*Aluminum foil tray, wafer tweezers, 2 Pyrex bowls and 2 lids |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="font-weight:bold;background:white">A. Making an SU-8 Master of Your
| + | |
- | Design</span></p>
| + | :'''Coating''' |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | #Carefully center your wafer on the spinner chuck. It may be difficult to tell if it’s properly positioned due to the white splatter guard. |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | #Pour approximately 5ml of SU-8(50) into a small plastic beaker. Pour as much as possible (~ 4ml) carefully in the CENTER of the wafer so as not to entrap air. |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Use the following</span></li>
| + | #Place the lid on the Spinner. Check the display to see if Program 001 is selected. Press the green Start button on the left of the spinner. The green light should turn on. |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">A pretreated
| + | ::*Program 1 yields a coating thickness of 100 mm: |
- | silicon wafer that has been cleaned with acetone and isopropyl alcohol
| + | :::Spread Cycle: 500rpm at 100rpm/sec for 5s |
- | (IPA) and dehydrated at 200</span><span style="vertical-align:super;
| + | :::Spin Cycle: 1200rpm at 300rpm/sec for 30 s. |
- | font-family:Arial;font-size:10.0pt;background:white">o</span><span style="font-family:Arial;font-size:10.0pt;background:white">C for 10-20
| + | #When the green light turns off, remove the spinner lid. Carefully lift your wafer off the chuck with your tweezers. |
- | minutes.</span></li>
| + | |
- | </ol>
| + | :'''Soft Bake''' |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | #Place it on the 65oC hot plate for 10 mins. After 10 mins increase the temp to 95oC (105C for VWR hot plates) with the wafer still on the hotplate and leave at this temp for 30 minutes. |
- | <li value="3" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | #While you are still baking your wafer |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Hot plate – make sure it is set to 65</span><span style="font-style: normal; vertical-align: super; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">o</span><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">C (80C for VWR hot plates)</span></li>
| + | ::*Collect your mask. |
- | </ol>
| + | ::*Clean off glass square with acetone, IPA, and then DI water. Dry with air gun – once clean only hold at edges and use only clean room paper towels |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="4" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | :'''Exposure''' |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Aluminum foil tray, wafer tweezers, 2 Pyrex bowls and 2 lids</span></li>
| + | #After 30 minutes at 95oC, let wafer cool on foil for 1 minute. Then take it over to the second clean room. PUT ON UV GLASSES. |
- | </ol>
| + | #Expose your wafer to UV light with your mask in the contact aligner. |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"> </p>
| + | #Brief Operating Instructions for the Contact Aligner: |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="font-weight:bold;background:white">Coating</span></p>
| + | ::*Check that vacuum pump is on in the LER |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | ::*Turn on Contact Aligner |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | ::*Press MASK – this lifts up Mask holder to place wafer |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Carefully </span><span style="font-family:Arial;font-size:
| + | ::*Load wafer, long flat side forward (facing away from you), and turn SUB ON to apply vacuum to hold wafer |
- | 10.0pt;font-weight:bold;font-style:normal;font-weight:bold;font-family:
| + | ::*Press MASK again – to lower mask holder |
- | Arial;font-size:10.0pt;background:white">center</span><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; "> your
| + | ::*Place mask printed (shiny side) down and bank it against the pins. Place glass cover on top. |
- | wafer on the spinner chuck. It may be difficult to tell if
| + | ::*Press MASK-ON to activate vacuum. If you hear the vacuum, you have not achieved a good seal. Check the mask. |
- | it’s properly positioned due to the white splatter guard. </span></li>
| + | ::*Raise the wafer to touch mask: Depress small round black button on left of chuck, while pressing this button, turn the gap micrometer Counter Clockwise. Once contact is made the gap dial will slip. |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Pour
| + | ::*Turn on the CONT. switch on the right panel. |
- | approximately 5ml of SU-8(50) into a small plastic
| + | ::*Set exposure time to 65 seconds (preset) |
- | beaker. Pour as much as possible (~ 4ml) carefully in the
| + | ::*Turn on the lamp power (white switch underneath the unit) and Start button |
- | CENTER of the wafer so as not to entrap air. </span></li>
| + | ::*CYCLE – the microscope unit will position itself over the mask. |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Place the lid
| + | ::*CYCLE – UV light will automatically expose over your sample. |
- | on the Spinner. Check the display to see if Program 001 is
| + | ::*CYCLE – UV source will move to home position. |
- | selected. Press the green Start button on the left of the
| + | ::*MASK – Mask holder will lift |
- | spinner. The green light should turn on. </span></li>
| + | ::*SUB-ON – wafer will be released from vacuum |
- | <ol type="a" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | ::*Remove your wafer and dial the gap micrometer clockwise to increase distance to mask. |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | ::*MASK to put down mask holder |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Program 1 yields a coating thickness of 100 mm:</span></li>
| + | ::*Press PULL OFF to release Mask vacuum. |
- | </ol>
| + | ::*Take off your mask |
- | </ol>
| + | |
- | <p style="margin:0in;margin-left:.75in;font-family:Arial;font-size:10.0pt;
| + | :'''Post Expose Bake''' |
- | color:black"><span style="background:white">Spread Cycle: 500rpm at
| + | #Perform a post bake of your wafer at 65oC (3rd hotplate) for 1 minute then 95oC for 10 mins to cross link the SU-8. Allow to cool for 1 minute. |
- | 100rpm/sec for 5s</span></p>
| + | |
- | <p style="margin:0in;margin-left:.75in;font-family:Arial;font-size:10.0pt;
| + | :'''Develop''' |
- | color:black"><span style="background:white">Spin Cycle: 1200rpm at
| + | #Place your exposed mask in the first Pyrex dish; fill the dish with SU-8 developer until the mask is covered and cover dish with the glass lid. Swish around for 8 mins. You should observe the removal of the SU-8 and see your design features. |
- | 300rpm/sec for 30 s.</span></p>
| + | #After 8 mins place your developed wafer in your second Pyrex container with fresh SU-8 developer, cover and swish for 2 mins. |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | #At this point some of your group members can leave the clean room to start making the PDMS in the mail lab area. |
- | <li value="4" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | #Pick up wafer, again holding it along the edge so you do not scratch the surface, with tweezers. Examine your wafer. Are their any undeveloped regions? If yes, place back into the developer solution and swish. The developing time depends on the complexity of the design. More complicated designs take longer to develop. |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">When the green light turns off, remove the spinner
| + | #If no hold it over the glass container with your tweezers and rinse with SU-8 developer, then with IPA and finally dry thoroughly with the air gun under the hood. A good drying technique is to aim and hold the gun directly in the center of the wafer. Be careful, gun can be at a high pressure resulting in you dropping your wafer and having to start from the beginning again. |
- | lid. Carefully lift your wafer off the chuck with your
| + | #Place your clean wafer into your labeled case and take it outside to the PDMS station. |
- | tweezers.</span></li>
| + | #Those remaining in the clean room must clean up! All solutions must go into the assigned waste container. Rinse glass containers with soap and water in the sink. Set them to dry on clean room paper towels. Remove undeveloped SU-8 50 on your tweezers by wiping it with developer. |
- | </ol>
| + | |
- | <p style="margin:0in;margin-left:.375in;font-family:Arial;font-size:10.0pt;
| + | ==Making PDMS== |
- | color:black"> </p>
| + | ''This section is performed in the BME Lab outside the clean room. Safety glasses, lab coats and gloves are required.'' |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="font-weight:bold;background:white">Soft Bake</span></p>
| + | #Weigh out 5g of hardener (small bottle) in a plastic cup, next add 50 g of base (large tub) to make the PDMS. A 1:10 ratio (by weight) of hardener: base is the recommended ratio that should be used. Typically, 55g of PDMS is sufficient to cover the bottom of the standard Petri plate that you will use. |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | #Beat the base/hardener mixture with a plastic fork (like beating an egg) vigorously until completely mixed and aerated (at least 5 minutes), the mixture should be white with lots of air bubbles. To be safe, beat the PDMS until your lab group comes out with the wafer. |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | #Secure your master wafer onto your Petri dish with some silicone glue and place in the 60C oven until PDMS is well aerated. |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Place it on the 65</span><span style="font-style: normal; vertical-align: super; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">o</span><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">C hot plate
| + | #Pour your PDMS mixture in the center of your wafer for an even coat. Place your dish in the vacuum chamber to remove air bubbles. |
- | for 10 mins. After 10 mins increase the temp to 95</span><span style="font-style: normal; vertical-align: super; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">o</span><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">C (105C for VWR hot plates) with the wafer still on the
| + | #Turn on the vacuum pump and watch your Petri dish. |
- | hotplate and leave at this temp for 30 minutes.</span></li>
| + | ::*When the bubble mixture looks like it will overflow, stop the pump. |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">While you are
| + | ::*Close the inlet/outlet by turning the black valve. Unhook the clear vacuum tubing. |
- | still baking your wafer </span></li>
| + | ::*Open the inlet/outlet and let the air out. |
- | <ol type="a" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | ::*Restart the vacuum pump and repeat these steps (~3-4x) until all bubbles are gone. |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | ::*This should take you ~15 minutes. |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Collect your mask.</span></li>
| + | #Make sure your dishes and lids are labeled. |
- | </ol>
| + | #bake at 60°C overnight to cure the PDMS. |
- | <ol type="a" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="2" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | :::''The top (as it sits in the Petri dish) of your PDMS micromixer device is called the “viewing side” while the bottom (the side with the channel indentations) is termed the “channel side.” The Channel Side Must Be Kept Clean At ALL Times!!'' |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Clean off glass square with</span><span style="font-family:Arial;
| + | |
- | font-size:10.0pt;font-weight:bold;font-style:normal;font-weight:bold;
| + | ==Sealing Device== |
- | font-family:Arial;font-size:10.0pt;background:white"> </span><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">acetone,
| + | #Clean glass slide with soap, acetone, IPA, then with water. Dry with air gun (located in on first bench). |
- | IPA, and then DI water. Dry with air gun – once clean only
| + | #Using the Xacto knife/razor cut out around your device with enough room around the inlets and outlet to make a good seal |
- | hold at edges and use only clean room paper towels</span></li>
| + | ::Make sure the channel side is even, with no irregularities caused by the PDMS molding around the tape or wafer edges. |
- | </ol>
| + | #Place your PDMS device in the clean square Petri dish – channel side down. |
- | </ol>
| + | #Using the purple needles, punch holes in your inlets and outlets. Firmly push down on the needle and gently pull out of the PDMS. Use the long yellow needles to push out the PDMS plug that should be now stuck in the barrel of the purple needle. |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="font-weight:bold;background:white">Exposure</span></p>
| + | ::Make Sure the PDMS Plug has been Removed. If there is no plug in the barrel, it means your channel has not been punched through fully. Use the yellow needle to push the plug out of the device. |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | :'''Plasma Treatment:''' |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">After 30 minutes at 95</span><span style="font-style: normal; vertical-align: super; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">o</span><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">C, let wafer
| + | ''To get the PDMS to stick to either glass or silicon, you must treat the two surfaces with oxygen plasma to create reactive groups for bonding.'' |
- | cool on foil for 1 minute. Then take it over to the second clean
| + | #Place your device with the CHANNEL SIDE FACING UP and the binding surface – a clean glass slide into the plasma cleaner/sterilizer. Make sure that at least one other group is ready to use the machine. |
- | room. </span><span style="font-family:Arial;font-size:10.0pt;
| + | #Close the pressure valve (black knob) on the door. Turn on the vacuum pump for approximately 5 minutes to evacuate the chamber of the sterilizer. |
- | font-weight:bold;font-style:normal;font-weight:bold;font-family:Arial;
| + | #After 5 minutes, turn on the power switch (the setting dial should be on “high”). This turns on the RF coil and should ignite the plasma in about 30 sec. |
- | font-size:10.0pt;background:white">PUT ON UV GLASSES.</span></li>
| + | #When the plasma ignites (you can tell by the purple glow you seen in the chamber window), start timing for 1 minute. |
- | </ol>
| + | #After 1 minute, turn off the power switch, and then the vacuum switch and slowly open the valve on the door to allow air to enter the chamber. |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | #THIS STEP IS TIME SENSITIVE: when you have taken your samples, move quickly out of the way for the next group! |
- | <li value="2" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | ::*When the pressure has normalized, remove your samples from the chamber and IMMEDIATELY invert the CHANNEL SIDE of your device onto the glass/silicon surface that was face up in the chamber. |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Expose your wafer to UV light with your mask in the contact
| + | ::*Gently press down on the device to ensure contact between the two surfaces. You should be able to initially see edge effects between portions that are in contact and those that are not. |
- | aligner.</span></li>
| + | #If you realize that a part of your channel is uneven – inhibiting proper contact, Quickly use the Xacto knife to remove uneven edges. |
- | </ol>
| + | #Bake for at least 15 min. at 95°C on a hot plate to promote bonding between the surfaces. |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | #After 15-25 minutes, take your device off the hot plate, and allow to cool for a minute. |
- | <li value="3" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | #Insert tubing in inlets and outlet. |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Brief Operating Instructions for the Contact Aligner:</span></li>
| + | |
- | <ul type="disc" style="margin-left:.375in;direction:ltr;unicode-bidi:embed;
| + | ==Streptavidin Coating and Fluorescent Testing== |
- | margin-top:0in;margin-bottom:0in">
| + | ''*All Needles and Syringes must be disposed of in the Sharps Container*'' |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Check that
| + | #Cut tubing and insert into device at the inflow and outflow ports. |
- | vacuum pump is on in the LER</span></li>
| + | #Using a syringe, fill the device with the following chemicals and incubate for the corresponding times. Remember to use different syringes for different chemicals. When inserting the chemicals, gently push down on the syringe with you thumb. |
- | </ul>
| + | #45 min in 4% (by volume) MPTMS in ethanol |
- | <ul type="disc" style="margin-left:.375in;direction:ltr;unicode-bidi:embed;
| + | #20 min in 1mM GMBS |
- | margin-top:0in;margin-bottom:0in">
| + | #45 min in 25ng/mL NeutrAvidin in PBS |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Turn on
| + | #Fill device with DI water and store in 4 degree fridge. |
- | Contact Aligner</span></li>
| + | #If you are adding ATTO520 (http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Datasheet/6/77810dat.Par.0001.File.tmp/77810dat.pdf), fill device with the solution for 20min at 5ul/min. Use a Harvard PHD 2000 syringe pump and cover the entire setup with aluminum foil so that the fluorescent probe is exposed to as little light as possible. |
- | </ul>
| + | #Flush the device with a syringe filled with air, then take pictures. |
- | <ul type="disc" style="margin-left:.375in;direction:ltr;unicode-bidi:embed;
| + | #Store in DI water in 4 degree fridge in the dark. |
- | margin-top:0in;margin-bottom:0in">
| + | :''Protocol is a standard chemistry procedure from Gleghorn et al: http://www.ncbi.nlm.nih.gov/pubmed/20024046'' |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Press MASK –
| + | ::''Reference was generously provided by Dr. Kirby (Cornell University, Mechanical and Aerospace Engineering)'' |
- | this lifts up Mask holder to place wafer</span></li>
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Load wafer,
| + | |
- | long flat side forward (facing away from you), and turn SUB ON to apply
| + | |
- | vacuum to hold wafer</span></li>
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Press MASK
| + | |
- | again </span><span style="font-weight:bold;font-family:Arial;
| + | |
- | font-size:10.0pt;background:white">–</span><span style="font-family:
| + | |
- | Arial;font-size:10.0pt;background:white"> to lower mask holder</span></li>
| + | |
- | </ul>
| + | |
- | <ul type="disc" style="margin-left:.375in;direction:ltr;unicode-bidi:embed;
| + | |
- | margin-top:0in;margin-bottom:0in">
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Place mask
| + | |
- | printed (shiny side) down and bank it against the pins. Place
| + | |
- | glass cover on top.</span></li>
| + | |
- | </ul>
| + | |
- | <ul type="disc" style="margin-left:.375in;direction:ltr;unicode-bidi:embed;
| + | |
- | margin-top:0in;margin-bottom:0in">
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Press
| + | |
- | MASK-ON to activate vacuum. If you hear the vacuum, you have not
| + | |
- | achieved a good seal. Check the mask.</span></li>
| + | |
- | </ul>
| + | |
- | <ul type="disc" style="margin-left:.375in;direction:ltr;unicode-bidi:embed;
| + | |
- | margin-top:0in;margin-bottom:0in">
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Raise the
| + | |
- | wafer to touch mask: Depress small round black button on left
| + | |
- | of chuck, while pressing this button, turn the gap micrometer Counter
| + | |
- | Clockwise. Once contact is made the gap dial will slip.</span></li>
| + | |
- | </ul>
| + | |
- | <ul type="disc" style="margin-left:.375in;direction:ltr;unicode-bidi:embed;
| + | |
- | margin-top:0in;margin-bottom:0in">
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Turn on the
| + | |
- | CONT. switch on the right panel.</span></li>
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Set exposure
| + | |
- | time to 65 seconds (preset)</span></li>
| + | |
- | </ul>
| + | |
- | <ul type="disc" style="margin-left:.375in;direction:ltr;unicode-bidi:embed;
| + | |
- | margin-top:0in;margin-bottom:0in">
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Turn on the
| + | |
- | lamp power (white switch underneath the unit) and Start button</span></li>
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">CYCLE – the
| + | |
- | microscope unit will position itself over the mask.</span></li>
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">CYCLE – UV
| + | |
- | light will automatically expose over your sample.</span></li>
| + | |
- | </ul>
| + | |
- | <ul type="disc" style="margin-left:.375in;direction:ltr;unicode-bidi:embed;
| + | |
- | margin-top:0in;margin-bottom:0in">
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">CYCLE – UV
| + | |
- | source will move to home position.</span></li>
| + | |
- | </ul>
| + | |
- | <ul type="disc" style="margin-left:.375in;direction:ltr;unicode-bidi:embed;
| + | |
- | margin-top:0in;margin-bottom:0in">
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">MASK – Mask
| + | |
- | holder will lift</span></li>
| + | |
- | </ul>
| + | |
- | <ul type="disc" style="margin-left:.375in;direction:ltr;unicode-bidi:embed;
| + | |
- | margin-top:0in;margin-bottom:0in">
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">SUB-ON –
| + | |
- | wafer will be released from vacuum</span></li>
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Remove your
| + | |
- | wafer and dial the gap micrometer clockwise to increase distance to
| + | |
- | mask.</span></li>
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">MASK to put
| + | |
- | down mask holder</span></li>
| + | |
- | </ul>
| + | |
- | <ul type="disc" style="margin-left:.375in;direction:ltr;unicode-bidi:embed;
| + | |
- | margin-top:0in;margin-bottom:0in">
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Press PULL
| + | |
- | OFF to release Mask vacuum.</span></li>
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Take off
| + | |
- | your mask</span></li>
| + | |
- | </ul>
| + | |
- | </ol>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"> </p>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="font-weight:bold;background:white">Post Expose Bake</span></p>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Perform a post bake of your wafer at 65</span><span style="font-style: normal; vertical-align: super; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">o</span><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">C (3rd hotplate) for 1 minute then 95</span><span style="font-style: normal; vertical-align: super; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">o</span><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">C for 10 mins to cross link the SU-8. Allow
| + | |
- | to cool for 1 minute.</span></li>
| + | |
- | </ol>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"> </p>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="font-weight:bold;background:white">Develop</span></p>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Place your exposed mask in the first Pyrex dish; fill the dish
| + | |
- | with SU-8 developer until the mask is covered and cover dish with the
| + | |
- | glass lid. Swish around for 8 mins. You should observe the
| + | |
- | removal of the SU-8 and see your design features.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="2" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">After 8 mins place your developed wafer in your second Pyrex
| + | |
- | container with fresh SU-8 developer, cover and swish for 2 mins.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="3" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">At this point some of your group members can leave the clean room
| + | |
- | to start making the PDMS in the mail lab area.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="4" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Pick up wafer, again holding it along the edge so you do not
| + | |
- | scratch the surface, with tweezers. Examine your
| + | |
- | wafer. Are their any undeveloped regions? If yes,
| + | |
- | place back into the developer solution and swish. The developing time
| + | |
- | depends on the complexity of the design. More complicated designs take
| + | |
- | longer to develop.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="5" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">If no hold it over the glass container with your tweezers and
| + | |
- | rinse with SU-8 developer, then with IPA and finally dry thoroughly with
| + | |
- | the air gun under the hood. A good drying technique is to aim
| + | |
- | and hold the gun directly in the center of the wafer. Be
| + | |
- | careful, gun can be at a high pressure resulting in you dropping your
| + | |
- | wafer and having to start from the beginning again.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="6" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Place your clean wafer into your labeled case and take it outside
| + | |
- | to the PDMS station.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="7" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Those remaining in the clean room must clean up! All
| + | |
- | solutions must go into the assigned waste container. Rinse
| + | |
- | glass containers with soap and water in the sink. Set them to
| + | |
- | dry on clean room paper towels. Remove undeveloped SU-8 50 on your
| + | |
- | tweezers by wiping it with developer. </span></li>
| + | |
- | </ol>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"> </p>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="font-weight:bold;background:white">B. Making PDMS</span></p>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="background:white">This section is performed in the BME Lab outside the
| + | |
- | clean room. Safety glasses, lab coats and gloves are required.</span></p>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Weigh out 5g of hardener (small bottle) in a plastic cup, next add
| + | |
- | 50 g of base (large tub) to make the PDMS. A 1:10 ratio (by
| + | |
- | weight) of hardener: base is the recommended ratio that should be
| + | |
- | used. Typically, 55g of PDMS is sufficient to cover the bottom
| + | |
- | of the standard Petri plate that you will use.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="2" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Beat the base/hardener mixture with a plastic fork (like beating
| + | |
- | an egg) vigorously until completely mixed and aerated (at least 5
| + | |
- | minutes), the mixture should be white with lots of air
| + | |
- | bubbles. To be safe, beat the PDMS until your lab group comes
| + | |
- | out with the wafer.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="3" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Secure your master wafer onto your Petri dish with some silicone
| + | |
- | glue and place in the 60C oven until PDMS is well aerated. </span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="4" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Pour your PDMS mixture in the center of your wafer for an even
| + | |
- | coat. Place your dish in the vacuum chamber to remove air
| + | |
- | bubbles. </span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="5" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Turn on the vacuum pump and watch your Petri dish. </span></li>
| + | |
- | <ol type="a" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">When the bubble mixture looks like it will overflow, stop the
| + | |
- | pump. Close the inlet/outlet by turning the black valve.</span></li>
| + | |
- | </ol>
| + | |
- | </ol>
| + | |
- | <p style="margin:0in;margin-left:.75in;font-family:Arial;font-size:10.0pt;
| + | |
- | color:black"><span style="background:white">Unhook the clear vacuum tubing</span></p>
| + | |
- | <p style="margin:0in;margin-left:.75in;font-family:Arial;font-size:10.0pt;
| + | |
- | color:black"><span style="background:white">Open the inlet/outlet and let the
| + | |
- | air out</span></p>
| + | |
- | <p style="margin:0in;margin-left:.75in;font-family:Arial;font-size:10.0pt;
| + | |
- | color:black"><span style="background:white">Restart the vacuum pump and repeat
| + | |
- | these steps (~3-4x) until all bubbles are gone. This should take you ~15
| + | |
- | minutes (Just think, your other group members are cleaning up while you sit
| + | |
- | and watch bubbles!)</span></p>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="6" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Make sure your dishes and lids are labeled. </span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="7" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">bake at 60°C overnight to cure the PDMS.</span></li>
| + | |
- | </ol>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="background:white"> </span></p>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="background:white">***The </span><span style="text-decoration:underline;
| + | |
- | background:white">top</span><span style="background:white"> (as it sits
| + | |
- | in the Petri dish) of your PDMS micromixer device is called the “viewing side”
| + | |
- | while the </span><span style="text-decoration:underline;background:white">bottom</span><span style="background:white"> (the side with the channel indentations) is
| + | |
- | termed the “channel side.” </span><span style="text-decoration:
| + | |
- | underline;background:white">The Channel Side Must Be Kept Clean At ALL Times!!</span></p> | + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="background:white"> </span></p>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="font-weight:bold;background:white">C. Sealing Device</span></p>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Clean glass slide with soap, acetone, IPA, then with
| + | |
- | water. Dry with air gun (located in on first bench).</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="2" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Using the Xacto knife/razor cut out around your device with enough
| + | |
- | room around the inlets and outlet to make a good seal</span></li>
| + | |
- | <ol type="a" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Make sure the channel side is even, with no irregularities caused
| + | |
- | by the PDMS molding around the tape or wafer edges.</span></li>
| + | |
- | </ol>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="3" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Place your PDMS device in the clean square Petri dish – channel
| + | |
- | side down. </span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="4" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Using the purple needles, punch holes in your inlets and
| + | |
- | outlets. Firmly push down on the needle and gently pull out of
| + | |
- | the PDMS. Use the long yellow needles to push out the PDMS
| + | |
- | plug that should be now stuck in the barrel of the purple needle.</span></li>
| + | |
- | <ol type="a" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Make Sure the PDMS Plug has been Removed. If there is no plug in
| + | |
- | the barrel, it means your channel has not been punched through fully.
| + | |
- | Use the yellow needle to push the plug out of the device.</span></li>
| + | |
- | </ol>
| + | |
- | </ol>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="background:white"> </span></p>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="font-weight:bold;background:white">D. Plasma Treatment: </span></p>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="background:white">To get the PDMS to stick to either glass or silicon,
| + | |
- | you must treat the two surfaces with oxygen plasma to create reactive groups
| + | |
- | for bonding.</span></p>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Place your device with the CHANNEL SIDE FACING UP and the binding
| + | |
- | surface – a </span><span style="font-family:Arial;font-size:10.0pt;
| + | |
- | font-weight:bold;font-style:normal;font-weight:bold;font-family:Arial;
| + | |
- | font-size:10.0pt;background:white">clean</span><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; "> glass slide into the
| + | |
- | plasma cleaner/sterilizer. Make sure that at least one other
| + | |
- | group is ready to use the machine. </span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="2" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Close the pressure valve (black knob) on the door. Turn
| + | |
- | on the vacuum pump for approximately 5 minutes to evacuate the chamber of
| + | |
- | the sterilizer. </span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="3" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">After 5 minutes, turn on the power switch (the setting dial should
| + | |
- | be on “high”). This turns on the RF coil and should ignite the
| + | |
- | plasma in about 30 sec.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="4" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">When the plasma ignites (you can tell by the purple glow you seen
| + | |
- | in the chamber window), start timing for 1 minute.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="5" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">After 1 minute, turn off the power switch, and then the vacuum
| + | |
- | switch and slowly open the valve on the door to allow air to enter the
| + | |
- | chamber.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="6" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">THIS STEP IS TIME SENSITIVE: when you have taken your
| + | |
- | samples, move quickly out of the way for the next group!</span></li>
| + | |
- | <ol type="a" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">When the pressure has normalized, remove your samples from the
| + | |
- | chamber and IMMEDIATELY invert the CHANNEL SIDE of your device onto the
| + | |
- | glass/silicon surface that was face up in the chamber. </span></li>
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">Gently press
| + | |
- | down on the device to ensure contact between the two
| + | |
- | surfaces. You should be able to initially see edge effects
| + | |
- | between portions that are in contact and those that are not.</span></li>
| + | |
- | </ol>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="7" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">If you realize that a part of your channel is uneven – inhibiting
| + | |
- | proper contact, Quickly use the Xacto knife to remove uneven
| + | |
- | edges. </span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="8" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Bake for </span><span style="font-style: italic; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">at least </span><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">15 min. at
| + | |
- | 95°C on a hot plate to promote bonding between the surfaces. </span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="9" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">After 15-25 minutes, take your device off the hot plate, and allow
| + | |
- | to cool for a minute. </span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="10" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Insert tubing in inlets and outlet.</span></li>
| + | |
- | </ol>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="background:white"> </span></p>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="font-weight:bold;background:white">E. Streptavidin Coating and
| + | |
- | Fluorescent Testing of the Microfluidic device.</span></p>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="1" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Cut tubing and insert into device at the inflow and outflow ports.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="2" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Using a syringe, fill the device with the following chemicals and
| + | |
- | incubate for the corresponding times. Remember to use different syringes
| + | |
- | for different chemicals. When inserting the chemicals, gently push down
| + | |
- | on the syringe with you thumb.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="3" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">45 min in 4% (by volume) MPTMS in ethanol</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="4" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">20 min in 1mM GMBS</span></li>
| + | |
- | <li style="margin-top:0;margin-bottom:0;vertical-align:middle;color:black"><span style="font-family:Arial;font-size:10.0pt;background:white">45 min in
| + | |
- | 25ng/mL NeutrAvidin in PBS</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="6" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Fill device with DI water and store in 4 degree fridge.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="7" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; color: black; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">If you are adding ATTO520 (</span><a href="http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Datasheet/6/77810dat.Par.0001.File.tmp/77810dat.pdf"><span style="font-family:Arial;font-size:10.0pt;font-family:Arial;font-size:
| + | |
- | 10.0pt;background:white">http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Datasheet/6/77810dat.Par.0001.File.tmp/77810dat.pdf</span></a><span style="font-style: normal; font-family: Arial; font-size: 10pt; color: black; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">),
| + | |
- | fill device with the solution for 20min at 5ul/min. Use a Harvard PHD
| + | |
- | 2000 syringe pump and cover the entire setup with aluminum foil so that
| + | |
- | the fluorescent probe is exposed to as little light as possible.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="8" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Flush the device with a syringe filled with air, then take
| + | |
- | pictures.</span></li>
| + | |
- | </ol>
| + | |
- | <ol type="1" style="margin-left: 0.375in; direction: ltr; unicode-bidi: embed; margin-top: 0in; margin-bottom: 0in; font-family: Arial; font-size: 10pt; font-style: normal; ">
| + | |
- | <li value="9" style="margin-top:0;margin-bottom:0;vertical-align:middle;
| + | |
- | color:black"><span style="font-style: normal; font-family: Arial; font-size: 10pt; background-image: initial; background-attachment: initial; background-origin: initial; background-clip: initial; background-color: white; background-position: initial initial; background-repeat: initial initial; ">Store in DI water in 4 degree fridge in the dark.</span></li>
| + | |
- | </ol>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="font-weight:bold;background:white"> </span></p>
| + | |
- | <p style="margin:0in;font-family:Arial;font-size:10.0pt;color:black"><span style="font-weight:bold;background:white">* All Needles and Syringes must be
| + | |
- | disposed of in the Sharps Container*</span></p></ul></b></p></ul>
| + | |
| | | |
- | </div>
| + | ==Cell Lysis== |
| + | #Spin cells down at 13000rpm for 10 min at 19 degrees celsius |
| + | #Remove supernatant and measure the mass of the pellet |
| + | #Add 2.5ml of Bugbuster Master for every 1 gram of cells |
| + | #Pipet up and down until the cells are well mixed, then shake for 20min |
| + | #Spin solution down again at 13000rpm for 10 min at 19 degrees celsius. |
| + | #Collect supernatant for experimental use |
| | | |
- | </html>
| + | ==Filtration== |
| + | #For use with PD-10 Desalting Columns from GE Healthcare |
| + | #Remove the top cap and pour off the column storage solution |
| + | #Remove the top filter using forceps |
| + | #Cut the sealed end of the column at notch |
| + | #Put the PD-10 Desalting column into a 50ml collection tube by suing the column adapter |
| + | #Fill up the column with 5ml of PBS and allow the PBS to enter the packed bed completely |
| + | #Repeat the above step 5 times |
| + | #Spin down at 1000rpm for 2min |
| + | #Put in lysate from cell lysis, spin down at 1000rpm for 2min |