Biomod/2012/Titech/Nano-Jugglers/Protocols: Difference between revisions
Line 86: | Line 86: | ||
# Resuspend micro-bieds pellet in 1 mL of Milli-Q water | # Resuspend micro-bieds pellet in 1 mL of Milli-Q water | ||
# Repeat steps 6-7 for five times | # Repeat steps 6-7 for five times | ||
# Resuspend micro-beads pellet in 20 µL of 10 µM thiol modified DNA and 10 mM | # Resuspend micro-beads pellet in 20 µL of 10 µM thiol modified DNA and 10 mM pH 8 Phosphate buffer | ||
# Incubate for 24 hours at room temperature with gentle mixing | # Incubate for 24 hours at room temperature with gentle mixing | ||
# Add 10 µL of 1 M NaCl, then Incubated at room temperature for 2 hours | # Add 10 µL of 1 M NaCl, then Incubated at room temperature for 2 hours | ||
Line 107: | Line 107: | ||
# Resuspend micro-bieds pellet in 1 mL of Milli-Q water | # Resuspend micro-bieds pellet in 1 mL of Milli-Q water | ||
# Repeat steps 6-7 for five times | # Repeat steps 6-7 for five times | ||
# Resuspend micro-beads pellet in 20 µL of 10 µM thiol modified DNA and 10 mM | # Resuspend micro-beads pellet in 20 µL of 10 µM thiol modified DNA and 10 mM pH 8 Phosphate buffer | ||
# Incubate for 24 hours at room temperature with gentle mixing | # Incubate for 24 hours at room temperature with gentle mixing | ||
# Add 10 µL of 1 M NaCl, then Incubated at room temperature for 2 hours | # Add 10 µL of 1 M NaCl, then Incubated at room temperature for 2 hours |
Revision as of 21:57, 27 October 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
- In this page, detailed protocols are described as supplementary information.
Vapor Deposition
- Preparation for deposition
- pipette 10 µL 10 µM polystyrene-beads into 2.0 mL tube.
- add 200 µL 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 1.0×10-3Pa)
- <energization>
- ※Au can't connect with polystyrene beads, so we deposit Au after Cr.
- 5. increase electric current slowly.
- 6. (Cr) 1st: stop at 15 A and keep 30 seconds. /2nd: stop at 15 A and keep 50 seconds.
- (Au): confirm melting at 8 A and keep 11 A until all Au has evaporated.
- 7. off electric current
- 8. cool down
- 9. increase pressure
- 10. take out the sample
- 11. vacuuming (to 1.0×101)
- ※※
- 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 2.0 mL 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
- 10 µm polystyrene beads, Au 2 cm, Cr, MilliQ, 70% ethanol
EDAC conjugation
- Pipet 12.5 mg of polystirene-beads into a 1.5 mL tube.
- Pellet the micro-beads via centrifugation for 5 minutes at 1000×G.
- 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 10 mg PolyLink EDAC in 50 µL 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×G.
- Resuspend micro-beads pellet in 0.4 mL Polylink Wash/Storage Buffer.
- Repeat Steps 12-13 for three times.
- Store particles at 4°C in Polylink Wash/Storage Buffer.
Confirmation of EDAC conjugation
- Pipet 12.5 mg of EDAC conjugated polystirene-beads into a 1.5 mL tube.
- Pellet the micro-beads via centrifugation for 5 minutes at 1000×G.
- Resuspend micro-beads pellet in 0.1 mL of 3×SSC Buffer
- Drip 5 µL of 10 µM Fluorescent beads with a complementary DNA
- Incubated for 10 minutes at 90℃
- Incubated for 40 minutes at room temperature
- Observing the fluorescence
Observation Conditions
- ISO6400
- Exposure time (Transmitted Light) 1/100 seconds
- Exposure time (Blue Light) 2 seconds
- Magnification 10×40=400
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 15 seconds at 1260-2680×G
- Remove platinum agglomerate
- Centrifuge mixture for 10 minutes at 15000×G
- Resuspend micro-beads in 0.4 mL of 1 M NaOH
- Incubate for 1 hour at room temperature with gentle mixing
- Centrifuge mixture for 10 minutes at 15000×G
- Resuspend micro-bieds pellet in 1 mL of Milli-Q water
- Repeat steps 6-7 for five times
- Resuspend micro-beads pellet in 20 µL of 10 µM thiol modified DNA and 10 mM pH 8 Phosphate buffer
- Incubate for 24 hours at room temperature with gentle mixing
- Add 10 µL of 1 M NaCl, then Incubated at room temperature for 2 hours
- Repeat steps 13 for 3 times.
- Centrifuge mixture for 10 minutes at 15000×G
- Resuspend micro-bieds pellet in 3×SSC buffer
- Repeat steps 15-16 for 3 times.
- Store particles at 4°C in 3×SSC buffer
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 15 seconds at 1260-2680×G
- Remove platinum agglomerate
- Centrifuge mixture for 10 minutes at 15000×G
- Resuspend micro-beads in 0.4 mL of 1 M NaOH
- Incubate for 1 hour at room temperature with gentle mixing
- Centrifuge mixture for 10 minutes at 15000×G
- Resuspend micro-bieds pellet in 1 mL of Milli-Q water
- Repeat steps 6-7 for five times
- Resuspend micro-beads pellet in 20 µL of 10 µM thiol modified DNA and 10 mM pH 8 Phosphate buffer
- Incubate for 24 hours at room temperature with gentle mixing
- Add 10 µL of 1 M NaCl, then Incubated at room temperature for 2 hours
- Repeat steps 13 for 3 times.
- Centrifuge mixture for 10 minutes at 15000×G
- Resuspend micro-bieds pellet in 3×SSC buffer
- Repeat steps 15-16 for 3 times.
- Store particles at 4°C in 3×SSC buffer
Catalyst conjugation
- Weight 1 mg of thiolated DNA modified platinum particles and DNA modified polystyrene beads into a 1.5 mL eppendorf tube
- Suspend micro-beads and platinum particles in 1 mL of 3×SSC buffer
- Incubate for 10 minutes at 90°C
- Incubate for 40 minutes at room temperature
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 µL and TEMED 4 µL.
- 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 1×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 (250 V or 300 V) for 20 minutes.
- Clear wells of the gel by pipette again.
- Load wells with sample solution 5 µL.(sample 3µL + 2×BPB solution 3 µL = sample solution 6 µL)
- Run at specified voltage (250 V or 300 V) for specified time (50 minutes)
- Put the gels into a SYBR gold solution taper. (SYBR-Gold solution: 10000×SYBR gold 20 µL + 1×TBE 200 mL)
- Shake the taper by hand for 10 minutes. (using used SYBR 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.
Ascertain that DNA form duplex in 1~5% H2O2 solution without degenerated by H2O2
- make sample
solution amount strength 40% acrylamide gel 5 mL 20% 10×TBE 1 mL 1× Milli-Q 4 mL Mass 10 mL
- 40% acrylamide gel 5 mL
- 10×TBE 1 mL
- MilliQ 4 mL
- 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 18 µL 16 µL 8 µL 10% H2O2 0 µL 0% 2 µL 1% 10 µL 5% 100 µM ssDNA F 0.5 µL 2.5 µM 0.5 µL 2.5 µM 0.5 µL 2.5 µM 100 µM ssDNA R 0.5 µL 2.5 µM 0.5 µL 2.5 µM 0.5 µL 2.5 µM 10×SSC 1 µL 0.5× 1 µL 0.5× 1 µL 0.5× Mass 20 µL 20 µL 20 µL
No. ④ ⑤ ⑥ ⑦ solution amount strengthen amount strengthen amount strengthen amount strengthen Milli-Q water 18.5 µL 8.5 µL 18.5 µL 8.5 µL 10% H2O2 0 µL 0% 10 µL 1% 0 µL 5% 10 100 µM ssDNA F 0.5 µL 2.5 µM 0.5 µL 2.5 µM 0 µL 0 µM 0 µL 0 µM 100 µM ssDNA R 0 µL 0 µM 0 µL 0 µM 0.5 µL 2.5 µM 0.5 µL 2.5 µM 10×SSC 1 µL 0.5× 1 µL 0.5× 1 µL 0.5× 1 µL 0.5× Mass 20 µL 20 µL 20 µL 20 µL
- 2. Incubate for 90 minutes at room temperature with gentle mixing.
- 3.anealing complementary strands for 10 minutes at 80℃
- 4. Incubate for 90 minutes at room temperature with gentle mixing.
- 5. Load wells with sample solution 5 µL. (sample 3 µL + 2×BPB solution 3 µL = sample solution 6 µL)
- 6. Run at specified voltage (250 V or 300 V) for specified time (50 minutes)
- 7. Put the gels into a SYBR gold solution taper. (SYBR-Gold solution: 10000×SYBR gold 20 µL + MilliQ water 200 mL)
- 8. Shake the taper by hand for 10 minutes.(using used SYBR 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.
Observation of the platinum particles
- Make a solvent of about 100 µL 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.
Observation of platinum hemisphere in solution of H2O2
- Weight 0.5 mg of platinum hemisphere particles into a schale
- Suspend 1~3% H2O2 solution until liquid surface is flat
- Wait 1 minute in order to stabilize bubble emissions
- Observed with a microscope
Observation of catalase conjugated 10 micro beads in solution of H2O2
- Weight 0.5 mg of catalase conjugated 10 micro beads into a schale
- Suspend 1~3% H2O2 solution until liquid surface is flat
- Wait 1 minute in order to stabilize bubble emissions
- Observed with a microscope
Observation of dissociation of dsDNA with UV-light
prepare
- 100 µM seqA 4Azo_5thiol←DNAi
- 100 µM seqAc 4Azo_5thiol←DNAii
- 2×SSC
- MilliQ water
solution A solution amount strength 100 µM seqA 4Azo_5thiol 5 µL 10 µM 2×SSC 25 µL 1× MlliQ water 20 µL Mass 50 µL
solution B solution amount strength 100 µM seqAc_5thiol 5 µL 10 µM 2×SSC 25 μL 1× MlliQ water 20 µL Mass 50 µL
Solution A+B solution amount strength A 50 µL DNAi 5 µM B 50 µL DNAii 5 µM Mass 100 µL
Finally prepared solution solution amount Solution A 10 µL Solution B 10 µL Solution A+B 80 µL
Way to hybridize DNA
- Irradiate visible-light (blue-light λ > 400 nm) to solution A and B for 5 minutes with Visible-light irradiation equipment (Epi-Green Slim pro S) to stabilize trans-azobenzene
- mix 40 µL of each solution under the visible light
- irradiate visible light for 10 minutes again
- Put solution into dark box at 4℃ for 12 hours to cool down for hybridization
Irradiate UV-light to DNA duplex and measures absorbance
- Irradiated UV-light (365 nm, 30 mW/cm2) to a solution A+B for 1, 5 minutes
- Measured absorbance of each DNA solutions (1~5) near 260 nm wavelength of light. The Abs of each DNA solution we measured was as follows (table below).
No Solusion (time exposed UV-light (MINUTES) ) 1 A (0) 2 B (0) 3 A+B (0) 4 A+B (1) 5 A+B (5)
- Irradiated UV-light (365 nm, 180mW/cm2) to a solution A+B for 5, 10, 30, 40, 50 seconds.
- Measured absorbance of each DNA solutions (1~8) near 260 nm wavelength of light. The Abs of each DNA solution we measured was as follows (table below).
No Solusion (time exposed UV-light (SECONDS) ) 1 A (0) 2 B (0) 3 A+B (0) 4 A+B (5) 5 A+B (10) 6 A+B (20) 7 A+B (30) 8 A+B (40) 9 A+B (50)
Material Lists and Kits
Name |
grade | Supplier |
Product code |
Cat No | Lot No |
Hydrogen peroxide (30%) | Wako 1st Grade | Wako | 081-04215 | *** | TLM1384 |
POLY BEAD CARBOXYLATE 10.0 micron microsperes | *** | Polyscience | 9003-53-6 | 18133 | 643610 |
Glass beads GBL-40 | *** | APPIE | 101021 | *** | JISZ890 |
Platinum 0.15~0.45 µm 99.9% | *** | ALDRICH | 1001302221 | 1310-73-2 | MKBH4608V |
Platinum 1 µm | *** | micromer | 01-83-103 | *** | 1551201-01 |
Catalase, from Bovine Liver | Wako 1st Grade | Wako | 039-12901 | EC1.11.1.6 | LAG1488 |
Sodium Dihidrogenphosphate Dihydrate | Wako 1st Grade | Wako | 192-02835 | *** | LAR4091 |
Disodium Hydrogenphosphate 12-Water | Wako 1st Grade | Wako | 196-02835 | *** | LAQ5931 |
6×Lording Buffer Double Dye | Wako 1st Grade | Wako | 313-90351 | *** | 02008D |
Sodium Chloride | Wako 1st Grade | Wako | 191-01665 | *** | DCN6646 |
Sodium Hydroxide | Wako 1st Grade | Wako | 198-13765 | 1310-73-2 | LAN1989 |
Ethnol(99.5) |
Wako 1st Grade |
Wako | 057-00451 | 64-17-5 | DBM6540 |
Albumin, from Bovine Serum, Cohn Fraction V, pH 7.0 |
Biochmistry |
Wako | 013-23291 | *** | STF3372 |
Ultra PureTM 1 M Tris-HCL pH 8.0 | *** | invitrogenTM | 15568-025 | *** | 9949164 |
40 (w/v) %-Acrylamide/Bis Mixed Solution (29:1) | SP | nacalai tesque | *** | *** | L1F7876 |
Sodium Dodecyl Sulfate (SDS) | Wako 1st Grade | Wako | 196-08675 | 151-4-3 | LAN1411 |
SSC Buffer 20×Concentrate | *** | SIGMA | *** | S6639-1L | 021M8403 |
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride | SU | TCI | D1601 | 25952-53-8 | FJXDI |
SYBR Gold nucleic acid gel stain | *** | life technologiesTM | S11494 | *** | 927072 |
Urea | for Molecular Biology | Wako | 211-01213 | 57-13-6 | LAQ5967 |
Tris-Borate-EDTA Buffer (10×), Nuclease an Protease tested [TBE Buffer] | *** | nacalai tesque | 35440-31 | *** | L1A6455 |
PolyLink - Protein Coupling Kit for COOH Microparticles For Microparticles 1.0 Micron or Larger | *** | Polysciences, Inc. | 24350 | *** | 631643 |
PolyLink EDAC | *** |
Polysciences, Inc. | 2435C | *** | 631321 |
Software
Sequence Design
- NUPACK: Software to design DNA arraignment.
- →Direct Link:http://www.nupack.org/partition/browser
- The DINAMelt Web Server: We used to know K_m of DNA strand.
- →Direct Link:http://mfold.rna.albany.edu/?q=DINAMelt
Software of editing
- Paint.net: Image processing software. We used it to control light and shade.
- →Direct Link:http://www.paint.net/
- Image J: Image analysis software. We used it to process photo of electrophoresis.
- →Direct Link:http://rsbweb.nih.gov/ij/
- Inkscape: We used it to draw figures.
- →Direct Link:http://inkscape.org/index.php?lang=en
- Chem Bio Draw: We used it mainly to draw chemical formula.
Software for YouTube
- Sakura editor: We used it to make music.
- →Direct Link:http://oto.chu.jp/
- Sound Engine: We used it to edit music.
- →Direct Link:http://soundengine.jp/