Biomod/2012/Titech/Nano-Jugglers/Protocols: Difference between revisions
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: In this page, detailed protocols are described as supplementary information. | : In this page, detailed protocols are described as supplementary information. | ||
:__TOC__ | :__TOC__ | ||
<h2>Vapor Deposition</h2> | <h2>Vapor Deposition</h2> | ||
*Preparation for deposition | *Preparation for deposition | ||
# pipette 10 | # pipette 10 µL 10 µM polystyrene-beads into 2.0 mL tube. | ||
# add | # add 200 µL milliQ into the tube. | ||
# mix the tube by vortex. | # mix the tube by vortex. | ||
# extend the polystyrene-beads on the cover glass. | # extend the polystyrene-beads on the cover glass. | ||
Line 16: | Line 17: | ||
# put target material (Cr, Au) in the basket. | # put target material (Cr, Au) in the basket. | ||
::※the amount of PVD is related to the Volume of targets. | ::※the amount of PVD is related to the Volume of targets. | ||
:4. vacuuming (to 1. | :4. vacuuming (to 1.0×10<sup><small>-3</small></sup>Pa) | ||
:<energization> | :<energization> | ||
::※Au can't connect with polystyrene beads, so we deposit Au after Cr. | ::※Au can't connect with polystyrene beads, so we deposit Au after Cr. | ||
:5. increase electric current slowly. | :5. increase electric current slowly. | ||
:6. (Cr) 1st: stop at | :6. (Cr) 1st: stop at 15 A and keep 30 seconds. / 2nd: stop at 15 A and keep 50 seconds. | ||
::(Au): confirm melting at | ::(Au): confirm melting at 8 A and keep 11 A until all Au has evaporated. | ||
:7. off electric current | :7. off electric current | ||
:8. cool down | :8. cool down | ||
:9. increase pressure | :9. increase pressure | ||
:10. take out the sample | :10. take out the sample | ||
:11. vacuuming(to 1. | :11. vacuuming (to 1.0×10<sup><small>1</small></sup>) | ||
::※※ | ::※※ | ||
# wash the surface of the cover glass with 70% ethanol. | # wash the surface of the cover glass with 70% ethanol. | ||
# 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. | # pipette 70% ethanol and polystyrene-beads into a 2.0 mL tube. | ||
# centrifuge the tube by tabletop centrifuge. | # centrifuge the tube by tabletop centrifuge. | ||
# remove supernatant. | # remove supernatant. | ||
Line 39: | Line 40: | ||
::Repeat※※ | ::Repeat※※ | ||
*Reagent | *Reagent | ||
: | :10 µm polystyrene beads, Au 2 cm, Cr, MilliQ, 70% ethanol | ||
<div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | <div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | ||
<h2>EDAC conjugation</h2> | <h2>EDAC conjugation</h2> | ||
# Pipet 12. | # Pipet 12.5 mg of polystirene-beads into a 1.5 mL tube. | ||
# Pellet the micro-beads via centrifugation for 5 minutes at | # Pellet the micro-beads via centrifugation for 5 minutes at 1000×G. | ||
# Resuspend micro-beads pellet in 0.4 | # Resuspend micro-beads pellet in 0.4 mL of PolyLink Coupling Buffer. | ||
# Pellet again via centrifugation for 5 minutes at | # Pellet again via centrifugation for 5 minutes at 1000×G. | ||
# Resuspend the micro-beads pellet in 0.17 | # Resuspend the micro-beads pellet in 0.17 mL of PolyLink Coupling Buffer. | ||
# Just before use, prepare a 200 mg/ | # Just before use, prepare a 200 mg/mL EDAC solution by dissolving 10 mg PolyLink EDAC in 50 µL Polylink Coupling Buffer. | ||
# Add 20 | # Add 20 µL of the EDAC solution to the micro-beads suspension. | ||
# Mix gently end-over-end or briefly vortex. | # Mix gently end-over-end or briefly vortex. | ||
# Add 5 nmol of aminated DNA. 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. | # Incubate for 90 minutes at room temperature with gentle mixing. | ||
# Centrifuge mixture for 10 minutes at | # Centrifuge mixture for 10 minutes at 1000×G. | ||
# Resuspend micro-beads pellet in 0. | # Resuspend micro-beads pellet in 0.4 mL Polylink Wash/Storage Buffer. | ||
# Repeat Steps 12-13 for three times. | # Repeat Steps 12-13 for three times. | ||
# Store particles at | # Store particles at 4°C in Polylink Wash/Storage Buffer. | ||
<div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | <div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | ||
<h2>Confirmation of EDAC conjugation</h2> | <h2>Confirmation of EDAC conjugation</h2> | ||
# Pipet 12. | # 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 | # Pellet the micro-beads via centrifugation for 5 minutes at 1000×G. | ||
# Resuspend micro-beads pellet in 0.1 | # Resuspend micro-beads pellet in 0.1 mL of 3×SSC Buffer | ||
# Drip | # Drip 5 µL of 10 µM Fluorescent beads with a complementary DNA | ||
# Incubated for 10 minutes at | # Incubated for 10 minutes at 90°C | ||
# Incubated for 40 minutes at room temperature | # Incubated for 40 minutes at room temperature | ||
# Observing the fluorescence | # Observing the fluorescence | ||
'''Observation Conditions''' | '''Observation Conditions''' | ||
:ISO6400<br> | :ISO6400<br> | ||
:Exposure | :Exposure time (Transmitted Light) 1/100 seconds<br> | ||
:Exposure | :Exposure time (Blue Light) 2 seconds<br> | ||
:Magnification 10×40=400<br> | :Magnification 10×40=400<br> | ||
<div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | <div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | ||
<h2>Gold and thiol modified DNA conjugation</h2> | <h2>Gold and thiol modified DNA conjugation</h2> | ||
# Weight 0.25 mg of gold-vapored 10 | # Weight 0.25 mg of gold-vapored 10 µm polystyrene beads into a 1.5 mL eppendorf tube. | ||
# Suspend micro-bieds in 1 | # Suspend micro-bieds in 1 mL of Milli-Q water | ||
# Pellet the micro-bieds via centrifugation for | # Pellet the micro-bieds via centrifugation for 15 seconds at 1260-2680×G | ||
# Remove platinum agglomerate | # Remove platinum agglomerate | ||
# Centrifuge mixture for 10 minutes at | # Centrifuge mixture for 10 minutes at 15000×G | ||
# Resuspend micro-beads in 0.4 | # Resuspend micro-beads in 0.4 mL of 1 M NaOH | ||
# Incubate for | # Incubate for 1 hour at room temperature with gentle mixing | ||
# Centrifuge mixture for 10 minutes at | # Centrifuge mixture for 10 minutes at 15000×G | ||
# Resuspend micro-bieds pellet in 1 | # 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 | # Resuspend micro-beads pellet in 20 µL of 10 µM thiol modified DNA and 10 mM pH 8 Phosphate buffer | ||
# Incubate for | # Incubate for 24 hours at room temperature with gentle mixing | ||
# Add | # Add 10 µL of 1 M NaCl, then Incubated at room temperature for 2 hours | ||
# Repeat steps 13 for 3 times. | # Repeat steps 13 for 3 times. | ||
# Centrifuge mixture for 10 minutes at | # Centrifuge mixture for 10 minutes at 15000×G | ||
# Resuspend micro-bieds pellet in | # Resuspend micro-bieds pellet in 3×SSC buffer | ||
# Repeat steps 15-16 for 3 times. | # Repeat steps 15-16 for 3 times. | ||
# Store particles at | # Store particles at 4°C in 3×SSC buffer | ||
<div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | <div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | ||
<h2>Platinum particle and thiol –modified DNA conjugation</h2> | <h2>Platinum particle and thiol –modified DNA conjugation</h2> | ||
# Weight 1 mg of | # Weight 1 mg of 0.15-0.40 µm platinum particle into a 1.5 mL eppendorf tube. | ||
# Suspend micro-bieds in 1 | # Suspend micro-bieds in 1 mL of Milli-Q water | ||
# Pellet the micro-bieds via centrifugation for | # Pellet the micro-bieds via centrifugation for 15 seconds at 1260-2680×G | ||
# Remove platinum agglomerate | # Remove platinum agglomerate | ||
# Centrifuge mixture for 10 minutes at | # Centrifuge mixture for 10 minutes at 15000×G | ||
# Resuspend micro-beads in 0.4 | # Resuspend micro-beads in 0.4 mL of 1 M NaOH | ||
# Incubate for | # Incubate for 1 hour at room temperature with gentle mixing | ||
# Centrifuge mixture for 10 minutes at | # Centrifuge mixture for 10 minutes at 15000×G | ||
# Resuspend micro-bieds pellet in 1 | # 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 | # Resuspend micro-beads pellet in 20 µL of 10 µM thiol modified DNA and 10 mM pH 8 Phosphate buffer | ||
# Incubate for | # Incubate for 24 hours at room temperature with gentle mixing | ||
# Add | # Add 10 µL of 1 M NaCl, then Incubated at room temperature for 2 hours | ||
# Repeat steps 13 for 3 times. | # Repeat steps 13 for 3 times. | ||
# Centrifuge mixture for 10 minutes at | # Centrifuge mixture for 10 minutes at 15000×G | ||
# Resuspend micro-bieds pellet in | # Resuspend micro-bieds pellet in 3×SSC buffer | ||
# Repeat steps 15-16 for 3 times. | # Repeat steps 15-16 for 3 times. | ||
# Store particles at | # Store particles at 4°C in 3×SSC buffer | ||
<div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | <div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | ||
<h2>Catalyst conjugation</h2> | <h2>Catalyst conjugation</h2> | ||
#Weight | #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 | #Suspend micro-beads and platinum particles in 1 mL of 3×SSC buffer | ||
#Incubate for 10 minutes at | #Incubate for 10 minutes at 90°C | ||
#Incubate for 40 minutes at room temperature | #Incubate for 40 minutes at room temperature | ||
<div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | <div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | ||
<h2>PAGE protocol</h2> | <h2>PAGE protocol</h2> | ||
# Make 20% polyacrylamide gel.<br> | # Make 20% polyacrylamide gel.<br> | ||
# Put into a microwave oven and liquefy urea for 15 seconds.<br> | # Put into a microwave oven and liquefy urea for 15 seconds.<br> | ||
# Wait until the solution become the room temperature.<br> | # Wait until the solution become the room temperature.<br> | ||
# Add 10% APS 150 | # Add 10% APS 150 µL and TEMED 4 µL.<br> | ||
# Put the solution into the gel box.<br> | # Put the solution into the gel box.<br> | ||
# Insert comb and wait it becomes coagulate(about 30 minutes).<br> | # Insert comb and wait it becomes coagulate (about 30 minutes).<br> | ||
# Make another gel box and prepare double gel boxes.<br> | # Make another gel box and prepare double gel boxes.<br> | ||
# Set up a tub for electrophoresis.<br> | # Set up a tub for electrophoresis.<br> | ||
# Set double gel boxes and pour | # 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).<br> | ||
# Incline the tub to remove bubbles under gels.<br> | # Incline the tub to remove bubbles under gels.<br> | ||
# Clear wells of the gel by pipette to remove unharden gel solution.<br> | # Clear wells of the gel by pipette to remove unharden gel solution.<br> | ||
# Connect the tub to a power source.<br> | # Connect the tub to a power source.<br> | ||
# Pre-run at specified voltage ( | # Pre-run at specified voltage (250 V or 300 V) for 20 minutes.<br> | ||
# Clear wells of the gel by pipette again.<br> | # Clear wells of the gel by pipette again.<br> | ||
# Load wells with sample solution | # Load wells with sample solution 5 µL.(sample 3µL + 2×BPB solution 3 µL = sample solution 6 µL)<br> | ||
# Run at specified voltage ( | # Run at specified voltage (250 V or 300 V) for specified time (50 minutes)<br> | ||
# Put the gels into a | # Put the gels into a SYBR gold solution taper. (SYBR-Gold solution: 10000×SYBR gold 20 µL + 1×TBE 200 mL)<br> | ||
# Shake the taper by hand for 10 minutes.(using used | # Shake the taper by hand for 10 minutes. (using used SYBR gold solution, shake for 15 or 20 minutes)<br> | ||
# Take out the gels.<br> | # Take out the gels.<br> | ||
# Spot blue light to observe the gel.<br> | # Spot blue light to observe the gel.<br> | ||
# Take pictures through an orange filter by camera.<br> | # Take pictures through an orange filter by camera.<br> | ||
<div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | <div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | ||
<h2>Ascertain that DNA form duplex in 1~5% | <h2>Ascertain that DNA form duplex in 1~5% H<sub>2</sub>O<sub>2</sub> solution without degenerated by H<sub>2</sub>O<sub>2</sub></h2> | ||
#make sample | #make sample | ||
:{| border="1" | :{| border="1" | ||
Line 147: | Line 154: | ||
|strength | |strength | ||
|- | |- | ||
|40%acrylamide gel | |40% acrylamide gel | ||
| | |5 mL | ||
|20% | |20% | ||
|- | |- | ||
| | |10×TBE | ||
| | |1 mL | ||
| | |1× | ||
|- | |- | ||
|Milli-Q | |Milli-Q | ||
| | |4 mL | ||
| | | | ||
|- | |- | ||
Line 166: | Line 173: | ||
*40% acrylamide gel | *40% acrylamide gel 5 mL | ||
* | *10×TBE 1 mL | ||
*MilliQ | *MilliQ 4 mL | ||
<br> | <br> | ||
*Way to make samples for electrophoresis<br> | *Way to make samples for electrophoresis<br> | ||
Line 190: | Line 197: | ||
|- | |- | ||
|Milli-Q water | |Milli-Q water | ||
| | |18 µL | ||
| | | | ||
| | |16 µL | ||
| | | | ||
| | |8 µL | ||
| | | | ||
|- | |- | ||
| | |10% H<sub>2</sub>O<sub>2</sub> | ||
| | |0 µL | ||
|0% | |0% | ||
| | |2 µL | ||
|1% | |1% | ||
| | |10 µL | ||
|5% | |5% | ||
|- | |- | ||
| | |100 µM ssDNA F | ||
|0. | |0.5 µL | ||
|2. | |2.5 µM | ||
|0. | |0.5 µL | ||
|2. | |2.5 µM | ||
|0. | |0.5 µL | ||
|2. | |2.5 µM | ||
|- | |- | ||
| | |100 µM ssDNA R | ||
|0. | |0.5 µL | ||
|2. | |2.5 µM | ||
|0. | |0.5 µL | ||
|2. | |2.5 µM | ||
|0. | |0.5 µL | ||
|2. | |2.5 µM | ||
|- | |- | ||
| | |10×SSC | ||
| | |1 µL | ||
|0. | |0.5× | ||
| | |1 µL | ||
|0. | |0.5× | ||
| | |1 µL | ||
|0. | |0.5× | ||
|- | |- | ||
|Mass | |Mass | ||
|20 µL | |20 µL | ||
| | | | ||
| | |20 µL | ||
| | | | ||
| | |20 µL | ||
| | | | ||
|} | |} | ||
Line 255: | Line 262: | ||
|- | |- | ||
|Milli-Q water | |Milli-Q water | ||
|18. | |18.5 µL | ||
| | | | ||
|8. | |8.5 µL | ||
| | | | ||
|18. | |18.5 µL | ||
| | | | ||
|8. | |8.5 µL | ||
| | | | ||
|- | |- | ||
| | |10% H<sub>2</sub>O<sub>2</sub> | ||
| | |0 µL | ||
|0% | |0% | ||
| | |10 µL | ||
|1% | |1% | ||
| | |0 µL | ||
|5% | |5% | ||
|10 | |10 | ||
| | | | ||
|- | |- | ||
| | |100 µM ssDNA F | ||
|0. | |0.5 µL | ||
|2. | |2.5 µM | ||
|0. | |0.5 µL | ||
|2. | |2.5 µM | ||
| | |0 µL | ||
| | |0 µM | ||
| | |0 µL | ||
| | |0 µM | ||
|- | |- | ||
| | |100 µM ssDNA R | ||
| | |0 µL | ||
| | |0 µM | ||
| | |0 µL | ||
| | |0 µM | ||
|0. | |0.5 µL | ||
|2. | |2.5 µM | ||
|0. | |0.5 µL | ||
|2. | |2.5 µM | ||
|- | |- | ||
| | |10×SSC | ||
| | |1 µL | ||
|0. | |0.5× | ||
| | |1 µL | ||
|0. | |0.5× | ||
| | |1 µL | ||
|0. | |0.5× | ||
| | |1 µL | ||
|0. | |0.5× | ||
|- | |- | ||
|<span style="color:red">Mass</span> | |<span style="color:red">Mass</span> | ||
|<span style="color:red"> | |<span style="color:red">20 µL</span> | ||
| | | | ||
|<span style="color:red"> | |<span style="color:red">20 µL</span> | ||
| | | | ||
|<span style="color:red"> | |<span style="color:red">20 µL</span> | ||
| | | | ||
|<span style="color:red"> | |<span style="color:red">20 µL</span> | ||
| | | | ||
|} | |} | ||
:2. Incubate for | :2. Incubate for 90 minutes at room temperature with gentle mixing. | ||
:3.anealing complementary strands for 10 minutes at | :3.anealing complementary strands for 10 minutes at 80°C | ||
:4. Incubate for | :4. Incubate for 90 minutes at room temperature with gentle mixing. | ||
:5. Load wells with sample solution | :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 ( | :6. Run at specified voltage (250 V or 300 V) for specified time (50 minutes) | ||
:7. Put the gels into a | :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 | :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. | :9. Take out the gels. | ||
:10. Spot blue light to observe the gel. | :10. Spot blue light to observe the gel. | ||
Line 327: | Line 334: | ||
<hr> | <hr> | ||
<h2>Observation of the platinum particles</h2> | <h2>Observation of the platinum particles</h2> | ||
# Make a solvent of about | # Make a solvent of about 100 µL in an Eppendorf tube.<br> | ||
# Put beads in another Eppendorf tube with a spatula.<br> | # Put beads in another Eppendorf tube with a spatula.<br> | ||
# Remove the dust on the surface of silicon rubber with the like Scotch tape.<br> | # Remove the dust on the surface of silicon rubber with the like Scotch tape.<br> | ||
Line 338: | Line 345: | ||
# Observe with the eyepiece.<br> | # Observe with the eyepiece.<br> | ||
<div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | <div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | ||
<h2>Observation of platinum hemisphere in solution of H< | <h2>Observation of platinum hemisphere in solution of H<sub>2</sub>O<sub>2</sub></h2> | ||
#Weight 0.5 mg of platinum hemisphere particles into a schale | #Weight 0.5 mg of platinum hemisphere particles into a schale | ||
#Suspend 1~3% H< | #Suspend 1~3% H<sub>2</sub>O<sub>2</sub> solution until liquid surface is flat | ||
#Wait 1 minute in order to stabilize bubble emissions | #Wait 1 minute in order to stabilize bubble emissions | ||
#Observed with a microscope | #Observed with a microscope | ||
<div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | <div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | ||
<h2>Observation of catalase conjugated 10 micro beads in solution of H< | <h2>Observation of catalase conjugated 10 micro beads in solution of H<sub>2</sub>O<sub>2</sub></h2> | ||
#Weight 0.5 mg of catalase conjugated 10 micro beads into a schale | #Weight 0.5 mg of catalase conjugated 10 micro beads into a schale | ||
#Suspend 1~3% H< | #Suspend 1~3% H<sub>2</sub>O<sub>2</sub> solution until liquid surface is flat | ||
#Wait 1 minute in order to stabilize bubble emissions | #Wait 1 minute in order to stabilize bubble emissions | ||
#Observed with a microscope | #Observed with a microscope | ||
Line 352: | Line 359: | ||
<h2>Observation of dissociation of dsDNA with UV-light</h2> | <h2>Observation of dissociation of dsDNA with UV-light</h2> | ||
'''prepare'''<br> | '''prepare'''<br> | ||
* | *100 µM seqA 4Azo_5thiol<span style="color:red">←DNAi</span> | ||
* | *100 µM seqAc 4Azo_5thiol<span style="color:red">←DNAii</span> | ||
*2×SSC | *2×SSC | ||
*MilliQ water | *MilliQ water | ||
Line 364: | Line 371: | ||
|strength | |strength | ||
|- | |- | ||
| | |100 µM seqA 4Azo_5thiol | ||
| | |5 µL | ||
| | |10 µM | ||
|- | |- | ||
| | |2×SSC | ||
| | |25 µL | ||
| | |1× | ||
|- | |- | ||
|MlliQ water | |MlliQ water | ||
| | |20 µL | ||
| | | | ||
|- | |- | ||
|<span style="color:red">Mass</span> | |<span style="color:red">Mass</span> | ||
|<span style="color:red"> | |<span style="color:red">50 µL</span> | ||
|} | |} | ||
:{| border="1" | :{| border="1" | ||
Line 386: | Line 393: | ||
|strength | |strength | ||
|- | |- | ||
| | |100 µM seqAc_5thiol | ||
| | |5 µL | ||
| | |10 µM | ||
|- | |- | ||
| | |2×SSC | ||
| | |25 μL | ||
| | |1× | ||
|- | |- | ||
|MlliQ water | |MlliQ water | ||
| | |20 µL | ||
| | | | ||
|- | |- | ||
|<span style="color:red">Mass</span> | |<span style="color:red">Mass</span> | ||
||<span style="color:red"> | ||<span style="color:red">50 µL</span> | ||
| | | | ||
|} | |} | ||
Line 410: | Line 417: | ||
|- | |- | ||
|A | |A | ||
| | |50 µL | ||
|<span style="color:red">DNAi</span> | |<span style="color:red">DNAi</span> 5 µM | ||
|- | |- | ||
|B | |B | ||
| | |50 µL | ||
|<span style="color:red">DNAii</span> | |<span style="color:red">DNAii</span> 5 µM | ||
|- | |- | ||
|Mass | |Mass | ||
| | |100 µL | ||
| | | | ||
|} | |} | ||
Line 428: | Line 435: | ||
|- | |- | ||
|Solution A | |Solution A | ||
| | |10 µL | ||
|- | |- | ||
|Solution B | |Solution B | ||
| | |10 µL | ||
|- | |- | ||
|Solution A+B | |Solution A+B | ||
| | |80 µL | ||
|} | |} | ||
'''Way to hybridize DNA''' | '''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 | #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 | #mix 40 µL of each solution under the visible light | ||
#irradiate visible light for | #irradiate visible light for 10 minutes again | ||
#Put solution into dark box at | #Put solution into dark box at 4°C for 12 hours to cool down for hybridization | ||
'''Irradiate UV-light to DNA duplex and measures absorbance''' | '''Irradiate UV-light to DNA duplex and measures absorbance''' | ||
#Irradiated UV-light( | #Irradiated UV-light (365 nm, 30 mW/cm<sup>2</sup>) to a solution A+B for 1, 5 minutes | ||
#Measured absorbance of each DNA solutions(1~5) near | #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). | ||
:{| border="1" | :{| border="1" | ||
|No | |No | ||
|Solusion (time exposed UV-light(MINUTES)) | |Solusion (time exposed UV-light (MINUTES) ) | ||
|- | |- | ||
|1 | |1 | ||
|A(0) | |A (0) | ||
|- | |- | ||
|2 | |2 | ||
|B(0) | |B (0) | ||
|- | |- | ||
|3 | |3 | ||
|A+B(0) | |A+B (0) | ||
|- | |- | ||
|4 | |4 | ||
|A+B(1) | |A+B (1) | ||
|- | |- | ||
|5 | |5 | ||
|A+B(5) | |A+B (5) | ||
|} | |} | ||
#Irradiated UV-light( | #Irradiated UV-light (365 nm, 180mW/cm<sup>2</sup>) to a solution A+B for 5, 10, 30, 40, 50 seconds. | ||
#Measured absorbance of each DNA solutions(1~8) near | #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). | ||
:{| border="1" | :{| border="1" | ||
|No | |No | ||
|Solusion (time exposed UV-light(SECONDS)) | |Solusion (time exposed UV-light (SECONDS) ) | ||
|- | |- | ||
|1 | |1 | ||
|A(0) | |A (0) | ||
|- | |- | ||
|2 | |2 | ||
|B(0) | |B (0) | ||
|- | |- | ||
|3 | |3 | ||
|A+B(0) | |A+B (0) | ||
|- | |- | ||
|4 | |4 | ||
|A+B(5) | |A+B (5) | ||
|- | |- | ||
|5 | |5 | ||
|A+B(10) | |A+B (10) | ||
|- | |- | ||
|6 | |6 | ||
|A+B(20) | |A+B (20) | ||
|- | |- | ||
|7 | |7 | ||
|A+B(30) | |A+B (30) | ||
|- | |- | ||
|8 | |8 | ||
|A+B(40) | |A+B (40) | ||
|- | |- | ||
|9 | |9 | ||
|A+B(50) | |A+B (50) | ||
|} | |} | ||
Line 511: | Line 518: | ||
<td align="center"><strong>Lot No</strong></td> | <td align="center"><strong>Lot No</strong></td> | ||
</tr><tr><td align="center"><strong>Hydrogen peroxide(30%)</strong></td> | </tr><tr><td align="center"><strong>Hydrogen peroxide (30%)</strong></td> | ||
<td> Wako 1st Grade</td> | <td> Wako 1st Grade</td> | ||
<td> Wako</td> | <td> Wako</td> | ||
Line 518: | Line 525: | ||
<td><font color="#000000">TLM1384</font></td> | <td><font color="#000000">TLM1384</font></td> | ||
</tr><tr><td align="center"><strong>POLY BEAD CARBOXYLATE 10. | </tr><tr><td align="center"><strong>POLY BEAD CARBOXYLATE 10.0 micron microsperes</strong></td> | ||
<td> ***</td> | <td> ***</td> | ||
<td> Polyscience</td> | <td> Polyscience</td> | ||
Line 532: | Line 539: | ||
<td><font color="#000000">JISZ890</font></td> | <td><font color="#000000">JISZ890</font></td> | ||
</tr><tr><td align="center"><strong>Platinum 0.15~0.45 | </tr><tr><td align="center"><strong>Platinum 0.15~0.45 µm 99.9%</strong></td> | ||
<td> ***</td> | <td> ***</td> | ||
<td> ALDRICH</td> | <td> ALDRICH</td> | ||
Line 539: | Line 546: | ||
<td><font color="#000000">MKBH4608V</font></td> | <td><font color="#000000">MKBH4608V</font></td> | ||
</tr><tr><td align="center"><strong>Platinum 1 | </tr><tr><td align="center"><strong>Platinum 1 µm</strong></td> | ||
<td> ***</td> | <td> ***</td> | ||
<td> micromer</td> | <td> micromer</td> | ||
Line 595: | Line 602: | ||
<td><font color="#000000">DBM6540</font></td> | <td><font color="#000000">DBM6540</font></td> | ||
</tr><tr><td align="center"><strong>Albumin, from Bovine Serum, Cohn Fraction V, | </tr><tr><td align="center"><strong>Albumin, from Bovine Serum, Cohn Fraction V, pH 7.0<br /> | ||
</strong></td> | </strong></td> | ||
<td> Biochmistry<br /></td> | <td> Biochmistry<br /></td> | ||
Line 603: | Line 610: | ||
<td><font color="#000000">STF3372</font></td> | <td><font color="#000000">STF3372</font></td> | ||
</tr><tr><td align="center"><strong>Ultra PureTM | </tr><tr><td align="center"><strong>Ultra PureTM 1 M Tris-HCL pH 8.0</strong></td> | ||
<td> *** </td> | <td> *** </td> | ||
<td> invitrogen<sup>TM</sup> </td> | <td> invitrogen<sup>TM</sup> </td> | ||
Line 610: | Line 617: | ||
<td><font color="#000000">9949164</font></td> | <td><font color="#000000">9949164</font></td> | ||
</tr><tr><td align="center"><strong>40(w/v)%-Acrylamide/Bis Mixed Solution (29:1)</strong></td> | </tr><tr><td align="center"><strong>40 (w/v) %-Acrylamide/Bis Mixed Solution (29:1)</strong></td> | ||
<td> SP</td> | <td> SP</td> | ||
<td> nacalai tesque </td> | <td> nacalai tesque </td> | ||
Line 617: | Line 624: | ||
<td><font color="#000000">L1F7876</font></td> | <td><font color="#000000">L1F7876</font></td> | ||
</tr><tr><td align="center"><strong>Sodium Dodecyl Sulfate(SDS)</strong></td> | </tr><tr><td align="center"><strong>Sodium Dodecyl Sulfate (SDS)</strong></td> | ||
<td> Wako 1st Grade </td> | <td> Wako 1st Grade </td> | ||
<td> Wako</td> | <td> Wako</td> | ||
Line 624: | Line 631: | ||
<td><font color="#000000">LAN1411</font></td> | <td><font color="#000000">LAN1411</font></td> | ||
</tr><tr><td align="center"><strong>SSC Buffer | </tr><tr><td align="center"><strong>SSC Buffer 20×Concentrate</strong></td> | ||
<td> ***</td> | <td> ***</td> | ||
<td> SIGMA</td> | <td> SIGMA</td> | ||
Line 652: | Line 659: | ||
<td><font color="#000000">LAQ5967</font></td> | <td><font color="#000000">LAQ5967</font></td> | ||
</tr><tr><td align="center"><strong>Tris-Borate-EDTA Buffer ( | </tr><tr><td align="center"><strong>Tris-Borate-EDTA Buffer (10×), Nuclease an Protease tested [TBE Buffer]</strong></td> | ||
<td> ***</td> | <td> ***</td> | ||
<td> nacalai tesque</td> | <td> nacalai tesque</td> | ||
Line 674: | Line 681: | ||
</table> | </table> | ||
<div align = "right" style="padding-right: | <div align = "right" style="padding-right:20px">[[#TOP|↑Page Top]]</div> | ||
<hr> | <hr> | ||
Line 697: | Line 704: | ||
*Sound Engine: We used it to edit music. | *Sound Engine: We used it to edit music. | ||
:→Direct Link:http://soundengine.jp/ | :→Direct Link:http://soundengine.jp/ | ||
<div align = "right" style="padding-right:200px">[[#TOP|↑Page Top]]</div> |
Latest revision as of 22:02, 27 October 2012
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} </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°C
- 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°C
- 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°C 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/