Biomod/2012/Titech/Nano-Jugglers/Protocols: Difference between revisions
No edit summary |
No edit summary |
||
Line 29: | Line 29: | ||
# Take pictures through an orange filter by camera.<br> | # Take pictures through an orange filter by camera.<br> | ||
<div align = "right">[[#TOP|↑]]</div> | <div align = "right" style="padding-right:200px">[[#TOP|↑]]</div> | ||
<h2>Observation of the platinum particles</h2> | <h2>Observation of the platinum particles</h2> | ||
# Make a solvent of about 100μL in an Eppendorf tube.<br> | # Make a solvent of about 100μL in an Eppendorf tube.<br> | ||
Line 42: | Line 42: | ||
# Observe with the eyepiece.<br> | # Observe with the eyepiece.<br> | ||
<div align = "right">[[#TOP|↑]]</div> | <div align = "right" style="padding-right:200px">[[#TOP|↑]]</div> | ||
<h2>Gold and thiol modified DNA conjugation</h2> | <h2>Gold and thiol modified DNA conjugation</h2> | ||
Line 60: | Line 60: | ||
# Store particles at 4℃ in 99.5% EtOH | # Store particles at 4℃ in 99.5% EtOH | ||
<div align = "right">[[#TOP|↑]]</div> | <div align = "right" style="padding-right:200px">[[#TOP|↑]]</div> | ||
<h2>Confirmation of Self assembled monolayer </h2> | <h2>Confirmation of Self assembled monolayer </h2> | ||
#Providing a cover glass with gold deposition | #Providing a cover glass with gold deposition | ||
Line 74: | Line 74: | ||
#Observing the fluorescence | #Observing the fluorescence | ||
<div align = "right">[[#TOP|↑]]</div> | <div align = "right" style="padding-right:200px">[[#TOP|↑]]</div> | ||
<h2>Platinum particle and thiol –modified DNA conjugation</h2> | <h2>Platinum particle and thiol –modified DNA conjugation</h2> | ||
Line 94: | Line 94: | ||
# Store particles at 4℃ in 99.5% EtOH | # Store particles at 4℃ in 99.5% EtOH | ||
<div align = "right">[[#TOP|↑]]</div> | <div align = "right" style="padding-right:200px">[[#TOP|↑]]</div> | ||
<h2>EDAC conjugation</h2> | <h2>EDAC conjugation</h2> | ||
Line 112: | Line 112: | ||
# Store particles at 4˚C in Polylink Wash/Storage Buffer. | # Store particles at 4˚C in Polylink Wash/Storage Buffer. | ||
<div align = "right">[[#TOP|↑]]</div> | <div align = "right" style="padding-right:200px">[[#TOP|↑]]</div> | ||
<h2>Physical Vapor Deposition</h2> | <h2>Physical Vapor Deposition</h2> | ||
Line 156: | Line 156: | ||
:10μm polystyrene beads, Au 2cm, Cr, MilliQ, 70%ethanol | :10μm polystyrene beads, Au 2cm, Cr, MilliQ, 70%ethanol | ||
<div align = "right">[[#TOP|↑]]</div> | <div align = "right" style="padding-right:200px">[[#TOP|↑]]</div> | ||
<h2>Azobenzen Protocol</h2> | <h2>Azobenzen Protocol</h2> | ||
Line 292: | Line 292: | ||
|} | |} | ||
<div align = "right">[[#TOP|↑]]</div> | <div align = "right" style="padding-right:200px">[[#TOP|↑]]</div> | ||
<h2>Ascertain that DNA form duplex in 1~5% H₂O₂ solution without degenerated by H₂O₂</h2> | <h2>Ascertain that DNA form duplex in 1~5% H₂O₂ solution without degenerated by H₂O₂</h2> | ||
Revision as of 00:21, 25 September 2012
<html> <head> <style type="text/css"> /* ====================
主に全体に関わるCSS
==================== */ body.mediawiki {
font-size: 14px;
background-image:url(http://openwetware.org/images/2/2d/TNJback.png); background-repeat: repeat; font-family: Calibri, Verdana, helvetica, sans-serif; }
h1 { padding: 0px 20px 5px 20px; font-size: 34px; line-height: 40px; font-weight: bold; } h2 { padding: 20px 20px 5px 20px; font-size: 25px; color: #000000; text-decoration: none; font-weight: bold; } h2 a { color: #eb8300; } h3 { padding: 20px 20px 5px 20px; font-size: 20px; color: #000; font-decoration: none; font-weight: bold; } h1.firstHeading {
display: none;
}
p { text-align: justify; } a:link { color: #00a5ea; text-decoration: none } a:visited { color:#00a5ea; text-decoration: none } a:hover {
color: #eb8300;
text-decoration: none } a:active { color:#f29400; text-decoration: none } #content{ margin: 0px 0 0px 0; align: center; padding: 12px 12px 12px 12px; width: 946px; background-color: #ffff; border: 0; } #bodyContent{ width: 970px; margin: 150px auto 0 auto; align: center; background-color: #ffffff; } #globalWrapper{ margin: 20px 0 0 0; width:994px; background-color: #ffffff; margin-left: auto; margin-right: auto } #biomodlink{ position:absolute;top:0px; width:970px;height:20px; background-color: #ffffff; margin:3px auto 3px auto }
/* ====================
メニューの画像を変更できる部分 ==================== */
#header {
margin: 0 auto 0 auto; position:absolute; top:28px; width:968px;height:189px;
background-color: #FFFFFF; background-repeat: no-repeat;
background-image: url(http://openwetware.org/images/7/74/Title-LOGO6.jpg);
} /* ====================
以下、特殊なclassに適用される ==================== */
#navigation { position:absolute;
top:120px; width:970px; height:92px; background-repeat: no-repeat;
color: #0000FF; }
/* ====================
ここからプルダウン周辺のデザイン ==================== */
- menu * {
margin: 0; padding: 0; }
- menu {
position:absolute;
font-family: calibri, verdana, sans-serif;
font-color: #000000;
background-color: transparent; float:left; display: block; }
- menu ul {
position:relative;
float: left; text-align:center; list-style: none; }
- menu li {
background-color:yellow;
position:relative;
float:left;
width: 136px;
font-size: 23px;
font-weight: bold;
border:ridge 0px #FFFFFF; }
- menu li.ach {
background-color:yellow;
position:relative;
float:left;
width: 162px;
font-size: 23px;
font-weight: bold;
border:ridge 0px #FFFFFF; }
- menu li.sup {
background-color:yellow;
position:relative;
float:left;
width: 109px;
font-size: 17px;
font-weight: bold;
border:ridge 0px #FFFFFF; }
- menu li.none {
background-color:yellow;
position:relative;
float:left;
width: 152px;
font-size: 17px;
font-weight: bold;
border:ridge 0px #FFFFFF; }
- menu a {
color: #000000; display:block; text-decoration: none; }
- menu a:hover {
background-color:gold; }
- column-one { display:none; width:0px;}
.container{background-color: #ffffff; margin-top:0px} .OWWNBcpCurrentDateFilled {display: none;}
- footer{position: center; width: 994px}
@media screen {
body { background: #F5F5F5 0 0 no-repeat; /* changed default background */ }
} </style> </head> <BODY> <div id="biomodlink"> <<a href="http://openwetware.org/wiki/Biomod">BIOMOD</a>|<a href="http://openwetware.org/wiki/Biomod/2012">2012</a>|Titech Nano-Jugglers </div> <div id="header"> <div id="navigation"> <div id="menu"> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers"><br>Home<br><br></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Team/Students"><br>Team<br><br></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Project"><br>Project<br><br></a></li> <li><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Results">Results<br>&<br>Methods</a></font></li> <li class="ach"><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Achievements"><br>Achievements<br><br></a> <li class="sup"><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Protocols"><br>Suppl. Info.<br><br></a></li> <li class="none"><a href="http://openwetware.org/wiki/Biomod/2012/Titech/Nano-Jugglers/Acknowledgement"><br>Acknowledgements<br><br></a></li> </ul> </div> </div> </div> </BODY> </html>
Protocols
PAGE protocol
- Make 20% polyacrylamide gel.
- Put into a microwave oven and liquefy urea for 15 seconds.
- Wait until the solution become the room temperature.
- Add 10% APS 150 ul and TEMED 4ul.
- Put the solution into the gel box.
- Insert comb and wait it becomes coagulate(about 30 minutes).
- Make another gel box and prepare double gel boxes.
- Set up a tub for electrophoresis.
- Set double gel boxes and pour 1x TBE running buffer into the tub (upper tub’s buffer is new running buffer, and lower tub’s buffer is recycle buffer).
- Incline the tub to remove bubbles under gels.
- Clear wells of the gel by pipette to remove unharden gel solution.
- Connect the tub to a power source.
- Pre-run at specified voltage (250V or 300V) for 20 minutes.
- Clear wells of the gel by pipette again.
- Load wells with sample solution 5ul.(sample 3ul + 2x BPB solution 3ul = sample solution 6ul)
- Run at specified voltage (250V or 300V) for specified time (50 minutes)
- Put the gels into a cyber gold solution taper.(SYBR-Gold solution: 10000x cyber gold 20ul + 1×TBE 200ml)
- Shake the taper by hand for 10 minutes.(using used cyber gold solution, shake for 15 or 20 minutes)
- Take out the gels.
- Spot blue light to observe the gel.
- Take pictures through an orange filter by camera.
Observation of the platinum particles
- Make a solvent of about 100μL in an Eppendorf tube.
- Put beads in another Eppendorf tube with a spatula.
- Remove the dust on the surface of silicon rubber with the like Scotch tape.
- Drill holes in the silicon rubber with a belt punch.
- Press the holed silicon rubber to the surface of a dish.
- Add the beads in the tube to the solvent.
- Shake the tube by voltex and set in the Ultrasound device.
- Inject the beads solution into the hole of silicon rubber.
- Set the dish to the observation units after down the objective lens to the bottom.
- Observe with the eyepiece.
Gold and thiol modified DNA conjugation
- weight 0.25 mg of gold-vapored 10 μm polystyrene beads into a 1.5 ml eppendorf tube.
- Suspend micro-bieds in 1 ml of Milli-Q water
- Pellet the micro-bieds via centrifugation for 10minutes at 15000xG
- Resuspend micro-beads in 0.4 ml of 1M NaOH.
- Incubate for 1h at room temperature with gentle mixing.
- Centrifuge mixture for 10 minutes at 15000 xG
- Resuspend micro-bieds pellet in 1 ml of Milli-Q water
- Repeat steps 6-7 for five times.
- Resuspend micro-beads pellet in 45μl of 99.5% EtOH and 5μl of 100 μM thiol-modified DNA
- Incubate for 24h at room temperature with gentle mixing.
- Centrifuge mixture for 10 minutes at 15000 xG
- Resuspend micro-bieds pellet in 99.5% EtOH
- Repeat steps 11-12 for 3 times.
- Store particles at 4℃ in 99.5% EtOH
Confirmation of Self assembled monolayer
- Providing a cover glass with gold deposition
- Immersed cover glass in 1M NaOH solution for 1 hour
- Rinse NaOH with MilliQ water twice
- Drip 5μl of 10μM thiol modified DNA and 10mM pH8 Phosphate buffer
- Incubated for 24 hours at room temperature
- Add 2μl of 1M NaCl, then Incubated at room temperature for 2hours
- Repeat steps 5-6 for 3 times.
- Rinse with 3×ssc buffer twice
- Drip 5μl of 10μM Fluorescent beads with a complementary DNA
- Incubated for 20 minutes at room temperature
- Observing the fluorescence
Platinum particle and thiol –modified DNA conjugation
- weight 1 mg of 0.15-0.40 μm platinum particle into a 1.5 ml eppendorf tube.
- Suspend micro-bieds in 1 ml of Milli-Q water
- Pellet the micro-bieds via centrifugation for 15seconds at 1260-2680 xG
- Remove platinum agglomerate
- Centrifuge mixture for 10 minutes at 15000 xG
- Resuspend micro-beads in 0.4 ml of 1M NaOH.
- Incubate for 1h at room temperature with gentle mixing.
- Centrifuge mixture for 10 minutes at 15000 xG
- Resuspend micro-bieds pellet in 1 ml of Milli-Q water
- Repeat steps 6-7 for five times.
- Resuspend micro-beads pellet in 45μl of 99.5% EtOH and 5μl of 100 μM thiol-modified DNA
- Incubate for 24h at room temperature with gentle mixing.
- Centrifuge mixture for 10 minutes at 15000 xG
- Resuspend micro-bieds pellet in 99.5% EtOH
- Repeat steps 13-14 for 3 times.
- Store particles at 4℃ in 99.5% EtOH
EDAC conjugation
- Pipet 12.5mg of polystirene-beads into a 1.5mL tube.
- Pellet the micro-beads via centrifugation for 5 minutes at 1000 xG.
- Resuspend micro-beads pellet in 0.4 ml of PolyLink Coupling Buffer.
- Pellet again via centrifugation for 5 minutes at 1000 G.
- Resuspend the micro-beads pellet in 0.17 ml of PolyLink Coupling Buffer.
- Just before use, prepare a 200 mg/ml EDAC solution by dissolving 10mg PolyLink EDAC in 50µl Polylink Coupling Buffer.
- Add 20 µl of the EDAC solution to the micro-beads suspension.
- Mix gently end-over-end or briefly vortex.
- Add 5 nmol of aminated DNA. Mix gently end-over-end or briefly vortex.
- Incubate for 90 minutes at room temperature with gentle mixing.
- Centrifuge mixture for 10 minutes at 1000 x G.
- Resuspend micro-beads pellet in 0.4ml Polylink Wash/Storage Buffer.
- Repeat Steps 12-13 for three times.
- Store particles at 4˚C in Polylink Wash/Storage Buffer.
Physical Vapor Deposition
- Preparation for deposition
- pipette 10 μL 10μM polystyrene-beads into 2.0mL tube.
- add 200μL milliQ into the tube.
- mix the tube by vortex.
- extend the polystyrene-beads on the cover glass.
- dry the cover glass.
- 1st Deposition
- increase the pressure in Bell jar.
- put cover off and lay the jar.
- put target material (Cr, Au) in the basket.
- ※the amount of PVD is related to the Volume of targets.
- 4. vacuuming (to 1.0×〖10〗^(-3)Pa )
- <energization>
- ※Au can't connect with polystyrene beads, so we deposit Au after Cr.
- 5. increase electric current slowly.
- 6. (Cr) 1st: stop at 15A and keep 30 seconds. /2nd: stop at 15A and keep 50 seconds.
- (Au): confirm melting at 8A and keep 11A until all Au has evaporated.
- 7. off electric current
- 8. cool down
- 9. increase pressure
- 10. take out the sample
- 11. vacuuming(to 1.0×〖10〗^1)
- ※※
- wash the surface of the cover glass with 70% ethanol.
- wash the surface of the cover glass with 70% ethanol.
- pipette 70% ethanol and polystyrene-beads into a 2.0mL tube.
- centrifuge the tube by tabletop centrifuge.
- remove supernatant.
- repeat 3 to 5
- extend the polystyrene-beads over a cover glass.
- dry the cover glass.
- 2nd Deposition
- Repeat※※
- Reagent
- 10μm polystyrene beads, Au 2cm, Cr, MilliQ, 70%ethanol
Azobenzen Protocol
Preparation
Solution A
solution amount strength 100μM seqAazo4_5thiol 5μL 10μM 2xSSC 25μL 1x MlliQ water 20μL Mass 50μL
- 100μM seqAazo4_5thiol(DNAⅰ) 5μL
- 2xSSC 25μL
- MlliQ water 20μL
solution B
solution amount strength 100μM seqAc_5thiol 5μL 10μM 2xSSC 25μL 1x MlliQ water 20μL Mass 50μL
- 100μM seqAc_5thiol(DNAⅱ) 5μl
- 2xSSC 25μL
- MlliQ water 20μL
Solution A+B
solution amount strength A 30μL (DNAⅰ)5μM B 30μL (DNAⅱ)5μM Mass 60μL
- solution A 30μL
- solution B 30μL
Finally prepared solution
solution amount Solution A 20μL Solution B 20μL Solution A+B 60μL
Way to Anneal DNA
- Irradiate visible-light to solutions A, B and A+B with Visible-light irradiation equipment(Epi-Green Slim pro S) to stabilize trans-azobanzen.
- Soaked solutions A, B, and A+B in a hot water of 100 digrees for 10 minute.
- Left these solutions A, B, and A+B for 50 minute, irradiating them visible-light.
- Put solutions into darkroom for 60 minute to cool down for stabilizing DNA duplex
Irradiate UV-light to DNA duplex and measures absorbance
- Irradiated UV-light(365nm) to a solution A+B for 1, 5, 30 minute.
- Measured absorbance of each DNA solutions(1~6) near 260nm wavelength of light. The DNA solutions we measured was as follows(table below).
No Solusion (time exposed UV-light) 1 A(0) 2 B(0) 3 A+B(0) 4 A+B(1) 5 A+B(5) 6 A+B(30)
Ascertain that DNA form duplex in 1~5% H₂O₂ solution without degenerated by H₂O₂
Way to make samples
20% polyacrilamide gels
solution amount strength 40%acrylamide gel 5mL 20% 10x TBE 1mL 1x Milli-Q 4mL Mass 10 mL
- 40% acrylamide gel 5mL
- 10x TBE 1mL
- MilliQ 4mL
Way to make samples for electrophoresis
Make solutions ①~⑦ as table below
No. ① ① ② ② ③ ③ solution amount strengthen amount strengthen amount strengthen Milli-Q water 18µL 16µL 8µL 10%H₂O₂ 0µL 0% 2µL 1% 10µL 5% 100µM ssDNA F 0.5µL 2.5µM 0.5µL 2.5µM 0.5µL 2.5µM 100µM ssDNA R 0.5µL 2.5µM 0.5µL 2.5µM 0.5µL 2.5µM 10xSSC 1µL 0.5x 1µL 0.5x 1µL 0.5x Mass 20 µL 20µL 20µL
No. ④ ⑤ ⑥ ⑦ solution amount strengthen amount strengthen amount strengthen amount strengthen Milli-Q water 18.5µL 8.5µL 18.5µL 8.5µL 10%H₂O₂ 0µL 0% 10µL 1% 0µL 5% 10 100µM ssDNA F 0.5µL 2.5µM 0.5µL 2.5µM 0µL 0µM 0µL 0µM 100µM ssDNA R 0µL 0µM 0µL 0µM 0.5µL 2.5µM 0.5µL 2.5µM 10xSSC 1µL 0.5x 1µL 0.5x 1µL 0.5x 1µL 0.5x Mass 20μL 20μL 20μL 20μL
- 2. Incubate for 90minutes at room temperature with gentle mixing.
- 3.anealing complementary strands for 10 minutes at 80℃
- 4. Incubate for 90minutes at room temperature with gentle mixing.
- 5. Load wells with sample solution 5ul.(sample 3ul + 2x BPB solution 3ul = sample solution 6ul)
- 6. Run at specified voltage (250V or 300V) for specified time (50 minutes)
- 7. Put the gels into a cyber gold solution taper.(SYBR-Gold solution: 10000x cyber gold 20ul + MilliQ water 200ml)
- 8. Shake the taper by hand for 10 minutes.(using used cyber gold solution, shake for 15 or 20 minutes)
- 9. Take out the gels.
- 10. Spot blue light to observe the gel.
- 11. Take pictures through an orange filter by camera.