Biomod/2011/TeamJapan/Tokyo/Notebook/Protocols

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Protocols

Contents

PAGE protocol

  1. Make 20% polyacrylamide gel.
  2. Put into a microwave oven and liquefy urea.
  3. Wait until the solution become the room temperature.
  4. Add 10% APS 150 ul and TEMED 4ul.
  5. Put the solution into the gel box.
  6. Insert comb and wait it harden (about 30 minutes).
  7. Make another gel box and prepare double gel boxes.
  8. Set up a tub for electrophoresis.
  9. 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).
  10. Incline the tub to remove bubbles under gels.
  11. Clear wells of the gel by pipette to remove unharden gel solution.
  12. Connect the tub to a power source.
  13. Pre-run at specified voltage (250V or 300V) for 20 minutes.
  14. Clear wells of the gel by pipette again.
  15. Load wells with sample solution 5ul.(sample 3ul + 2x BPB solution 3ul = sample solution 6ul)
  16. Run at specified voltage (250V or 300V) for specified time (50 minutes)
  17. Put the gels into a cyber gold solution taper.(SYBR-Gold solution: 10000x cyber gold 20ul + MilliQ water 200ml)
  18. Shake the taper by hand for 10 minutes.(using used cyber gold solution, shake for 15 or 20 minutes)
  19. Take out the gels.
  20. Spot blue light to observe the gel.
  21. 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

  1. 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
  2. Prepare DNA ciliate solution and centrifuge (15000rpm, 1minute) to make precipitation (DNA ciliate solution)
  3. Vacuum the precipitation 1.5~3.0 ul by pipette and put into the solutions (A and B)
  4. Mass up solutions to 8.5 ul by MilliQ water
  5. Pour 2uM substrate DNA 1.5ul into the solutions to start reaction
  6. React for 10~15 hours
  7. Stop deoxyribozyme reaction by 250mM EDTA 2.5ul to reaction solutions
  8. Mix by vortex
  9. Put the tube onto heating block (80°C) for 1 minute to separate substrate DNA from deoxyribozime DNA
  10. Centrifuge (15000rpm,1minute) to make precipitation
  11. Put the tube onto heating block (80°C) for 1 minute to separate substrate DNA from deoxyribozime DNA
  12. Vacuum the supernatant 3ul by pipette and put into 0.6ul tubes for samples
  13. Finish making samples(sample A is enzyme active sample, sample B is enzyme inactive sample)

negative control and positive control

  • Positive control
  1. Making the following solution.
    MilliQ water 3ul
    5x SSC 2ul
    Deoxyribozyme DNA 1.5ul
    Substrate DNA 1.5ul
    50mM ZnSO4 2ul
  2. Mix by vortex
  3. Vacuum 3ul by pipette and put into 0.6ul tubes for samples
  4. Finish making positive control
  • Negative control
  1. Making the following solution.
    MilliQ water 5ul
    5x SSC 2ul
    Deoxyribozyme DNA 1.5ul
    Substrate DNA 1.5ul
  2. Mix by vortex
  3. Vacuum 3ul by pipette and put into 0.6ul tubes for samples
  4. Finish making negative control

How to make DNA ciliate by EDAC

We used "PolyLink Protein Coupling Kit for COOH Microspheres" in this protocol.
  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.
  4. Pellet again via centrifugation for 5-10 minutes at approximately 500-1000 G.
  5. Resuspend the micro-beads pellet in 0.17ml of Polylink Coupling Buffer.
  6. Just before use, prepare a 200mg/ml EDAC solution by dissolving 10mg Polylink EDAC in 50µl Polylink Coupling Buffer.
  7. Add 20µl of the EDAC solution to the micro-beads suspension.
  8. Mix gently end-over-end or briefly vortex.
  9. Add 5nmol of aminated DNA. Mix gently end-over-end or briefly vortex.
  10. Incubate for 30-60 minutes at room temperature with gentle mixing.
  11. Centrifuge mixture for 10 minutes at approximately 500-1000 x G.
  12. Resuspend micro-beads pellet in 0.4ml Polylink Wash/Storage Buffer.
  13. Repeat Steps 12-13.
  14. Store particles at 4˚C in Polylink Wash/Storage Buffer.


UV switching 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
  1. Make DNA solutions in 5xSSC and 80mM MgCl2 buffer.The density of DNA solutions is 0.9uM.How to make is written in #Note.
  2. 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)
  3. 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.
  4. Close the tube’s caps.
  5. Turn on the heating blocks at 95°C.
  6. Put tubes onto heating block for 2 minute. (don’t take out tubes from the heating block)
  7. Turn off the heating block and wait until the heating block becomes room temperature. It takes about 2 hour.
  8. Take out tubes from the heating block.
  9. Open the tube’s caps and spot visible light (no UV) to the solutions for 30 min again.
  10. 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.)
  11. Close the tube’s caps.
  12. Set an UV light (365nm) equipment and turn on. (To stabilize the light, wait for 3 min.)
  13. Open tube’s caps and put tubes under UV light. The distance from solutions to UV light is 5 cm.
  14. Turn off the room light.
  15. Take out the tubes after spotting light for specific time and close the tube’s caps
  16. Turn on the room light. The solutions are samples for electrophoresis.
  17. 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

Immobilization of single-stranded DNA on glass plate using microchannel

  1. Place a MAS coated glass plate in 5mM DSS solution for an hour.
  2. Wash thrice the glass plate in Milli-Q water, and dry it completely.
  3. PDMS mold to overlay on the glass plate.
  4. Use a micropippet to add 20uM DNA solution droplets to inlets of the microchannel.
  5. Satisfying solution in the microchannels by the passive pumping.
  6. Incubate for 2 hours at room temperature.
  7. Wash the glass plate in 0.2% SDS solution.
  8. Wash thrice the glass plate in Mili-Q water, and dry it completely.
  9. Place the glass plate in 3% BSA solution for 30 minutes.
  10. Wash thrice the glass plate in Milli-Q water, and dry it completely.

Note

  • 5mM DSS solution
5mM DSS
DMSO
  • 20uM DNA solution
1xPBS
20uM DNA
  • 0.2% SDS solution
0.2% SDS
Milli-Q
  • 3% BSA solution
3% BSA
0.1M Tris-HCl


DNA hybridization to the complementary DNA protcol

  1. Pipette the hybridization mix (5×SSC, 0.2%SDS, 10uM DNA) on the spot DNA immobilized on glass. (Hybridization mix pipetted 20ul per coverslip.)
  2. Carefully lay the coverslip face down on the top of the hybridization mix.
  3. Place the glass and moist Kimwipes into the Petri dish. Seal up Petri dish by using Parafilm.
  4. Hybridize for 1h.(45°C)
  5. Cool Petri dish for 30 min at room temp.
  6. Wash the glass into the 5×SSC buffer of about 5mm.
  7. Wash the glass into the 1×SSC buffer of about 5mm twice.
  8. Observe the glass filled 1×SSC buffer.

DNA hybridization to the beads immobilized complementary DNA protcol

  1. 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.)
  2. Carefully lay the coverslip face down on the top of the hybridization mix.
  3. Place the glass and moist Kimwipes into the Petri dish. Seal up Petri dish by using Parafilm.
  4. Hybridize for 1h.(45°C)
  5. Cool Petri dish for 30 min at room temp.
  6. Wash the glass into the hybridization buffer A (5×SSC, 3%BSA) of about 5mm.
  7. Wash the glass into the hybridization buffer B (1×SSC, 3%BSA) of about 5mm twice.
  8. Observe the glass filled hybridization buffer B (1×SSC, 3%BSA).

Micromachining for polyformaldehyde(acetal)-resin protocol

  1. Cut polyacetal-resin at the size of slide with an electric saw.
  2. 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. )
  3. Fix polyacetal-resin on the base of micro fine machining center.
  4. Attach the 1mm diameter endmill to micro fine machining center.
  5. Set X, Y, and Z origins by using Vpanel.
  6. Cut polyacetal-resin by using Vpanel if necessary.
  7. Do face milling. (Do face milling means flatten the polyacetal-resin by endmill)
  8. 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.
  9. Make micro-channel according to the drawing. The roughly shape of micro-channel is the Figure-1.
  10. After carving, remove polyacetal-resin by using isopropyl alcohol (IPA).
  11. Observe micro-channel by using an optical microscope.
Figure-1:The roughly shape of micro-channel
Figure-1:The roughly shape of micro-channel

PDMS Mold Preparation Protcol

  1. Before you begin this experimentation, you must sterilize the experiment stand.
  2. Prepare PDMS, curing agent, electronic weigh and plastic tube which volume is 50ml.
  3. Determine 1/10th measurement of the amount of PDMS used and pipette curing agent to the plastic tube.
  4. Mix PDMS and curing agent about 10 minutes.
  5. Try not to have almost all bubbles in the PDMS by using vacuum desiccators on 30 minutes to one hour.
  6. After you check no bubble, apply the PDMS to the sample mold. The sample mold is made by polyacetal.
  7. Try not to have any bubbles in the PDMS by using vacuum desiccators on 30mimutes again.
  8. Prepare to use heater. (Switch on a heater.)
  9. Bake PDMS on the heater for 1 hour at 75°C.
  10. After heating it, cool it to room temperature.
  11. Remove fully cured PDMS samples from heater
  12. Cut PDMS along micro channel.

Note

  • PDMS=Polydimethylsiloxane
  • PDMS and curing agent we used is Silpot 184

Material Lists and Kits


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



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.
→Direct Link:http://www.cambridgesoft.com/software/details/?fid=15&pid=226
  • 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.
→Direct Link:http://www.cyberlink.com/products/powerdirector/overview_en_US.html
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