Biomod/2011/TeamJapan/Tokyo/Notebook/Protocols
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<div id="navigation"> <div id="menu" style="position:static"> <ul> <li><a class="aMain" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo">Home</a></li> <li><a class="aTeam" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Team/Students">Team</a></li> <li><a class="aProject" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project">Project</a> <!-- <ul> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project">Overview</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/introduction">Introduction</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Model">Model</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Devices">Devices</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Modes">Modes</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results">Results</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Achievements">Achievements</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Future_works">Future works</a></li> </ul> --> <li><font color="#ffffff">Results</font> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Results">Experiments</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Simulations">Simulations</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Achievements/DNA_Devices">DNA Design</a></li> </ul></li> <!-- <li><a class="Simulation" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Simulations">Simulations</a></li> <li><a class="DNA design" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Achievements/DNA_Devices">DNA Designs</a></li> --> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Achievements">Achievements</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Project/Future_works">Future works</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Notebook/Protocols">Protocols</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Notebook/Lab.notebook">Notes</a></li> <li><a class="aNotebook" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Sponsors/">Sponsors</a></li> <li><a class="aSitemap" href="http://openwetware.org/wiki/Biomod/2011/TeamJapan/Tokyo/Sitemap">Sitemap</a></li>
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Protocols
PAGE protocol
- Make 20% polyacrylamide gel.
- Put into a microwave oven and liquefy urea.
- 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 harden (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 + MilliQ water 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.
Notes
- How to make samples is here.
- How to make BPB solution is here.
- There are two type 20% polyacrilamide gels.
- With urea type (Checking DNA ciliate)
- 40% acrylamide gel (acrylamide:bisacrylamide =29:1) 5ml
- 10x TBE 1ml
- Urea 4.8g ( =4ml,8M )
- Mass: about 10ml
- Without urea type (Checking UV switch)
- 40% acrylamide gel (acrylamide:bisacrylamide =29:1) 5ml
- 10x TBE 1ml
- MilliQ 4ml
- Mass: about 10ml
How to make samples for electrophoresis
DNA ciliate
- Make this solution A in the 0.6ml tube
50mM ZnSO4 2μL
5x SSC buffer 2ul
(To check the enzyme activity for deoxyribozyme, remember to make the following solution B because deoxyribozyme activity requires Zn2+.)
MilliQ water 2ul
5x SSC buffer 2ul - Prepare DNA ciliate solution and centrifuge (15000rpm, 1minute) to make precipitation (DNA ciliate solution)
- Vacuum the precipitation 1.5~3.0 ul by pipette and put into the solutions (A and B)
- Mass up solutions to 8.5 ul by MilliQ water
- Pour 2uM substrate DNA 1.5ul into the solutions to start reaction
- React for 10~15 hours
- Stop deoxyribozyme reaction by 250mM EDTA 2.5ul to reaction solutions
- Mix by vortex
- Put the tube onto heating block (80°C) for 1 minute to separate substrate DNA from deoxyribozime DNA
- Centrifuge (15000rpm,1minute) to make precipitation
- Put the tube onto heating block (80°C) for 1 minute to separate substrate DNA from deoxyribozime DNA
- Vacuum the supernatant 3ul by pipette and put into 0.6ul tubes for samples
- Finish making samples(sample A is enzyme active sample, sample B is enzyme inactive sample)
negative control and positive control
- Positive control
- Making the following solution.
MilliQ water 3ul
5x SSC 2ul
Deoxyribozyme DNA 1.5ul
Substrate DNA 1.5ul
50mM ZnSO4 2ul - Mix by vortex
- Vacuum 3ul by pipette and put into 0.6ul tubes for samples
- Finish making positive control
- Negative control
- Making the following solution.
MilliQ water 5ul
5x SSC 2ul
Deoxyribozyme DNA 1.5ul
Substrate DNA 1.5ul - Mix by vortex
- Vacuum 3ul by pipette and put into 0.6ul tubes for samples
- Finish making negative control
Fixation of NH2 labeled oligonucleotide DNA probes to polystyrene beads
1. Pipet 12.5mg of micro-beads into a 1.5mL tube. 2. Pellet the micro-beads via centrifugation for 5-10 minutes at approximately 500-1000 xG. 3. Resuspend micro-beads pellet in 0.4ml of Polylink Coupling Buffer. 5. Pellet again via centrifugation for 5-10 minutes at approximately 500-1000 G. 6. Resuspend the micro-beads pellet in 0.17ml of Polylink Coupling Buffer. 7. Just before use, prepare a 200mg/ml EDAC solution by dissolving 10mg Polylink EDAC in 50µl Polylink Coupling Buffer. 8. Add 20µl of the EDAC solution to the micro-beads suspension. 9. Mix gently end-over-end or briefly vortex. 10. Add 5nmol of aminated DNA. Mix gently end-over-end or briefly vortex. 11. Incubate for 30-60 minutes at room temperature with gentle mixing. 12. Centrifuge mixture for 10 minutes at approximately 500-1000 x G. 13. Resuspend micro-beads pellet in 0.4ml Polylink Wash/Storage Buffer. 14. Repeat Steps 12-13. 15. Store particles at 4˚C in Polylink Wash/Storage Buffer.
UV switch protocol
- UV switch protocol (To check by electrophoresis)
- A…DNA Devices
- B…block DNA
- D…deoxyribozyme DNA
- 5x SSC…sodium citrate 75mM
- This experiment should be in UV cut room to prevent isomerizes
- Make DNA solutions in 5xSSC and 80mM MgCl2 buffer.The density of DNA solutions is 0.9uM.How to make is written in #Note.
- Mix A and B in 0.6 ml tubes to make solution A+B. Mix A and D in 0.6 ml tubes to make solution A+D. To check the A+D’s loss of color by UV, prepare two A+D solution’s tube
Solution A+B…A 0.3uM and B 0.6uM (Mix A and B)
Solution A+D…A 0.225uM and D 0.225uM (Mix A, D, and 80mM Mg2+ in 5x SSC solution)
(To make controls, don’t mix all solutions) - Open the tube caps and spot visible light (no UV) to the solutions for 30 minute.The distance from solutions to light is 9 cm.
- Close the tube’s caps.
- Turn on the heating blocks at 95°C.
- Put tubes onto heating block for 2 minute. (don’t take out tubes from the heating block)
- Turn off the heating block and wait until the heating block becomes room temperature. It takes about 2 hour.
- Take out tubes from the heating block.
- Open the tube’s caps and spot visible light (no UV) to the solutions for 30 min again.
- Mix A + B and D in 0.6ul tube to make reaction solutions. (A 0.225uM, B 0.45uM, D 0.225uM) (To make controls, don’t mix all solutions.)
- Close the tube’s caps.
- Set an UV light (365nm) equipment and turn on. (To stabilize the light, wait for 3 min.)
- Open tube’s caps and put tubes under UV light. The distance from solutions to UV light is 5 cm.
- Turn off the room light.
- Take out the tubes after spotting light for specific time and close the tube’s caps
- Turn on the room light. The solutions are samples for electrophoresis.
- Do electrophoresis. The protocol for electrophoresis is here
Note
- DNA solutions (DNA’s density is 0.9uM)
- 100uM DNA (A or B or D) 1ul
- 1M MgCl2 9ul
- 10x SSC 56ul
- MilliQ water 45ul
- DNA’s solution in 5xSSC, Mg2+ 80mM 111ul
- Control solutions for electrophoresis
- 1. A 0.225uM
- 2. B 0.450uM
- 3. D 0.225uM
- 4. A + B (A 0.225uM + B 0.450uM)
- 5. A + D (A 0.225uM + D 0.225uM)
- 6. A + D (spotted UV)…to check the UV-switching-trap-DNA’s loss of color by UV
- 80mM Mg<spb>2+</spb> in 5x SSC solution is used for dilutions
DNA hybridization to the complementary DNA protcol
- Pipette the hybridization mix (5×SSC, 0.2%SDS, 10uM DNA) on the spot DNA immobilized on glass. (Hybridization mix pipetted 20ul per coverslip.)
- Carefully lay the coverslip face down on the top of the hybridization mix.
- Place the glass and moist Kimwipes into the Petri dish. Seal up Petri dish by using Parafilm.
- Hybridize for 1h.(45°C)
- Cool Petri dish for 30 min at room temp.
- Wash the glass into the 5×SSC buffer of about 5mm.
- Wash the glass into the 1×SSC buffer of about 5mm twice.
- Observe the glass filled 1×SSC buffer.
DNA hybridization to the beads immobilized complementary DNA protcol
- Pipette the hybridization mix (5×SSC, 3%BSA and about 10uM beads immobilized DNA) on the spot DNA immobilized on glass. (Hybridization mix pipetted 20ul per coverslip.)
- Carefully lay the coverslip face down on the top of the hybridization mix.
- Place the glass and moist Kimwipes into the Petri dish. Seal up Petri dish by using Parafilm.
- Hybridize for 1h.(45°C)
- Cool Petri dish for 30 min at room temp.
- Wash the glass into the hybridization buffer A (5×SSC, 3%BSA) of about 5mm.
- Wash the glass into the hybridization buffer B (1×SSC, 3%BSA) of about 5mm twice.
- Observe the glass filled hybridization buffer B (1×SSC, 3%BSA).
Micromachining for polyformaldehyde(acetal)-resin protocol
- Cut polyacetal-resin at the size of slide with an electric saw.
- Boot up the micro fine machining center.(Model Number: Roland MODELA MDX-40A)
Unlock the emergency button.
Push the power button.
Get the computer to start up which is attached to micro fine machining center.
Execute the software of ClickMill and Vpanel.(ClickMill is the software which designates micro-channel’s shape. And Vpanel is the software which set the position of the endmill. ) - Fix polyacetal-resin on the base of micro fine machining center.
- Attach the 1mm diameter endmill to micro fine machining center.
- Set X, Y, and Z origins by using Vpanel.
- Cut polyacetal-resin by using Vpanel if necessary.
- Do face milling. (Do face milling means flatten the polyacetal-resin by endmill)
- Change the endmill which adjusts to the micro-channel’s width.(If micro-channel’s width is more than 10um, you don’t need to change endmill. If micro-channel’s width is between 50 um and 100 um, you should change to the endmill which diameter is 0.5um. Don’t make micro channel whose width is less than 50 um because it is difficult to carve exactly.
- Make micro-channel according to the drawing. The roughly shape of micro-channel is the Figure-1.
- After carving, remove polyacetal-resin by using isopropyl alcohol (IPA).
- Observe micro-channel by using an optical microscope.
PDMS Mold Preparation Protcol
- Before you begin this experimentation, you must sterilize the experiment stand.
- Prepare PDMS, curing agent, electronic weigh and plastic tube which volume is 50ml.
- Determine 1/10th measurement of the amount of PDMS used and pipette curing agent to the plastic tube.
- Mix PDMS and curing agent about 10 minutes.
- Try not to have almost all bubbles in the PDMS by using vacuum desiccators on 30 minutes to one hour.
- After you check no bubble, apply the PDMS to the sample mold. The sample mold is made by polyacetal.
- Try not to have any bubbles in the PDMS by using vacuum desiccators on 30mimutes again.
- Prepare to use heater. (Switch on a heater.)
- Bake PDMS on the heater for 1 hour at 75°C.
- After heating it, cool it to room temperature.
- Remove fully cured PDMS samples from heater
- Cut PDMS along micro channel.
Note
- PDMS=Polydimethylsiloxane
- PDMS and curing agent we used is Silpot 184
Material Lists and Kits
Laboratory instrument
- Micro fine machining center
- Roland MODELA MDX-40A
- http://www.rolanddg.com/product/3d/3d/mdx-40a/index.html
- End mill
- TECNOS Co, Ltd.
- -RSE230-0.5X2.5---Diameter is 0.5mm
- -RSE230-1X5---Diameter is 1mm
- -RSE230-3X15---Diameter is 3mm
- Mili-Q water
- Tabletop Centrifuge
- [1](Only Japanese)
- Ultracentrifuge
- HITACHI himac CT 15RE
- http://www.hitachi-koki.com/himac/centrifuges/ct15e/ct15e.html
- Fluorescence Phase contrast microscope
- OLYMPUS CKX41
- http://www.microscopy.olympus.eu/microscopes/Life_Science_Microscopes_Routine_microscope_CKX41.html
- Shaker
- Rotator of micro tube
- As One MTR-103
- http://www.justis.as-1.co.jp/jus-tis/web/DetailEnglish.aspx?sid=justis&catalog=GJ&group=G025401&no=&op_from=jus-tis
- Band Saw
- DESICCATOR VACUUM
- AS ONE 1-068-01
- http://www.justis.as-1.co.jp/jus-tis/web/DetailEnglish.aspx?sid=justis&catalog=GJ&group=G007103&no=&op_from=jus-tis
- Vacuum pumps
- TGK LV-435A
- http://www.tgk.co.jp/info/0904645105s.html
- Camera
- CASIO EX-F1
- http://www.casio-intl.com/dc/ex_f1/
- Heating Block
- THERMO BLOCK ND-M01
- http://www.tech-jam.com/scientific_research_equipment/incubator/kn3316700.phtml (Only Japanese)
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.
- aviutl: We used it mainly to edit YouTube videos.
- →Direct Link:http://www.gigafree.net/media/me/aviutl.html
- trakAxPC: We used it mainly to edit YouTube videos.
- →Direct Link:http://www.trakax.com/software/pc/download/
- CyberLink power director: We used it mainly to edit YouTube videos.
Name |
grade | Supplier |
Product code |
Cat No | Lot No |
Sodium Hydroxide | Wako 1st Grade | Wako | 198-13765 | 1310-73-2 | LAN1989 |
SILPOT 184 | *** |
DOW CORNING TORAY | 03255981 | *** | 0006410158 |
Acetone | Wako 1st Grade |
Wako | 012-00343 | 67-64-1 | DCN6798 |
2-Propanol |
Wako 1st Grade |
Wako | 166-04831 | 67-63-0 | DCM6848 |
Ethnol(99.5) |
Wako 1st Grade |
Wako | 057-00451 | 64-17-5 | DBM6540 |
Albumin, from Bovine Serum, Cohn Fraction V, pH7.0 |
Biochmistry |
Wako | 013-23291 | *** | STF3372 |
Di(N-succinimidyl) Suberate |
*** |
TCI | D3895 | 68528-80-3 | CU3BM |
Ultra PureTM 1M Tris-HCL pH 8.0 | *** | invitrogenTM | 15568-025 | *** | 9949164 |
40(w/v)%-Acrylamide/Bis Mixed Solution (29:1) | SP | nacalai tesque | *** | *** | L1F7876 |
Dimethyl Sulfoxide | Wako 1st Grade | Wako | 043-07216 | 67-68-5 | LAL4398 |
Sodium Dodecyl Sulfate(SDS) | Wako 1st Grade | Wako | 196-08675 | 151-4-3 | LAN1411 |
SSC Buffer 20x Concentrate | *** | SIGMA | *** | S6639-1L | 021M8403 |
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride | SU | TCI | D1601 | 25952-53-8 | FJXDI |
N-Hydroxysuccinimide | *** | TCI | H0623 | 6066-82-6 | EOC3E |
Suberic acid bis(3-sulfo-N-hydroxysuccinimide ester) sodium salt | *** | SIGMA | S5799-25MG | *** | 051M4067V |
SYBR Gold nucleic acid gel stain | *** | life technologiesTM | S11494 | *** | 927072 |
Dimethyl Sulfoxide | for Molecular Biology | Wako | 041-29351 | 67-68-5 | DCN0023 |
Zinc Sulfate | Practical Grade | Wako | 268-00422 | 7733-02-0 | LAQ6174 |
Urea | for Molecular Biology | Wako | 211-01213 | 57-13-6 | LAQ5967 |
Magnesium Chloride | *** | Wako | 136-03995 | 7786-30-3 | LAR4676 |
Tris-Borate-EDTA Buffer (10X), Nuclease an Protease tested [TBE Buffer] | *** | nacalai tesque | 35440-31 | *** | L1A6455 |
Ammonium Peroxodisulfate | Wako 1st Grade | Wako | 018-03282 | 7727-54-0 | HLH7635 |
N,N,N',N'-TETRAMETHYL- ETHYLENEDIAMINE | Wako 1st Grade | Wako | 202-04003 | 110-18-9 | EPF1167 |
0.5N EDTA (pH8.0) | *** | Wako | 311-90075 | *** | 01481B |
Glycerol | Wako 1st Grade | Wako | 075-00616 | 56-81-5 | LAQ5416 |
Disodium Hydrogenphosphate 12-Water | Wako 1st Grade | Wako | 196-02835 | 10039-32-4 | LAQ5931 |
Sodium Dihydrogenphosphate Dihydrate | Wako 1st Grade | Wako | 192-02815 | 13472-35-0 | LAR4091 |
Hydrochloride Acid | Wako 1st Grade | Wako | 080-01066 | 7647-01-0 | LAN1065 |
PolyLink - Protein Coupling Kit for COOH Microparticles For Microparticles 1.0 Micron or Larger | *** | Polysciences, Inc. | 24350 | *** | 631643 |
PolyLink EDAC | *** |
Polysciences, Inc. | 2435C | *** | 631321 |