Biomod/2012/Titech/Nano-Jugglers/Protocols

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Line 4: Line 4:
:__TOC__
:__TOC__
-
<h2>Physical Vapor Deposition</h2>
+
<h2>Vapor Deposition</h2>
*Preparation for deposition
*Preparation for deposition

Revision as of 20:46, 13 October 2012


Protocols

Contents


Vapor Deposition

  • Preparation for deposition
  1. pipette 10 μL 10μM polystyrene-beads into 2.0mL tube.
  2. add 200μL milliQ into the tube.
  3. mix the tube by vortex.
  4. extend the polystyrene-beads on the cover glass.
  5. dry the cover glass.
  • 1st Deposition
  1. increase the pressure in Bell jar.
  2. put cover off and lay the jar.
  3. put target material (Cr, Au) in the basket.
※the amount of PVD is related to the Volume of targets.
4. vacuuming (to 1.0×〖10〗^(-3)Pa )
<energization>
※Au can't connect with polystyrene beads, so we deposit Au after Cr.
5. increase electric current slowly.
6. (Cr) 1st: stop at 15A and keep 30 seconds. /2nd: stop at 15A and keep 50 seconds.
(Au): confirm melting at 8A and keep 11A until all Au has evaporated.
7. off electric current
8. cool down
9. increase pressure
10. take out the sample
11. vacuuming(to 1.0×〖10〗^1)
※※
  1. wash the surface of the cover glass with 70% ethanol.
  2. wash the surface of the cover glass with 70% ethanol.
  3. pipette 70% ethanol and polystyrene-beads into a 2.0mL tube.
  4. centrifuge the tube by tabletop centrifuge.
  5. remove supernatant.
  6. repeat 3 to 5
  7. extend the polystyrene-beads over a cover glass.
  8. dry the cover glass.
  • 2nd Deposition
Repeat※※
  • Reagent
10μm polystyrene beads, Au 2cm, Cr, MilliQ, 70%ethanol

Gold and thiol modified DNA conjugation

  1. Weight 0.25 mg of gold-vapored 10 μm polystyrene beads into a 1.5 ml eppendorf tube.
  2. Suspend micro-bieds in 1 ml of Milli-Q water
  3. Pellet the micro-bieds via centrifugation for 15seconds at 1260-2680 xG
  4. Remove platinum agglomerate
  5. Centrifuge mixture for 10 minutes at 15000 xG
  6. Resuspend micro-beads in 0.4 ml of 1M NaOH
  7. Incubate for 1h at room temperature with gentle mixing
  8. Centrifuge mixture for 10 minutes at 15000 xG
  9. Resuspend micro-bieds pellet in 1 ml of Milli-Q water
  10. Repeat steps 6-7 for five times
  11. Resuspend micro-beads pellet in 20μl of 10μM thiol modified DNA and 10mM pH8 Phosphate buffer
  12. Incubate for 24h at room temperature with gentle mixing
  13. Add 10μl of 1M NaCl, then Incubated at room temperature for 2hours
  14. Repeat steps 13 for 3 times.
  15. Centrifuge mixture for 10 minutes at 15000 xG
  16. Resuspend micro-bieds pellet in 3×ssc buffer
  17. Repeat steps 15-16 for 3 times.
  18. Store particles at 4℃ in 3×ssc buffer


Confirmation of Self assembled monolayer

  1. Providing a cover glass with gold deposition
  2. Immersed cover glass in 1M NaOH solution for 1 hour
  3. Rinse NaOH with MilliQ water twice
  4. Drip 5μl of 10μM thiol modified DNA and 10mM pH8 Phosphate buffer
  5. Incubated for 24 hours at room temperature
  6. Add 2μl of 1M NaCl, then Incubated at room temperature for 2hours
  7. Repeat steps 6 for 3 times.
  8. Rinse with 3×ssc buffer twice
  9. Drip 5μl of 10μM Fluorescent beads with a complementary DNA
  10. Incubated for 20 minutes at room temperature
  11. Observing the fluorescence

Platinum particle and thiol –modified DNA conjugation

  1. Weight 1 mg of 0.15-0.40 μm platinum particle into a 1.5 ml eppendorf tube.
  2. Suspend micro-bieds in 1 ml of Milli-Q water
  3. Pellet the micro-bieds via centrifugation for 15seconds at 1260-2680 xG
  4. Remove platinum agglomerate
  5. Centrifuge mixture for 10 minutes at 15000 xG
  6. Resuspend micro-beads in 0.4 ml of 1M NaOH
  7. Incubate for 1h at room temperature with gentle mixing
  8. Centrifuge mixture for 10 minutes at 15000 xG
  9. Resuspend micro-bieds pellet in 1 ml of Milli-Q water
  10. Repeat steps 6-7 for five times
  11. Resuspend micro-beads pellet in 20μl of 10μM thiol modified DNA and 10mM pH8 Phosphate buffer
  12. Incubate for 24h at room temperature with gentle mixing
  13. Add 10μl of 1M NaCl, then Incubated at room temperature for 2hours
  14. Repeat steps 13 for 3 times.
  15. Centrifuge mixture for 10 minutes at 15000 xG
  16. Resuspend micro-bieds pellet in 3×ssc buffer
  17. Repeat steps 15-16 for 3 times.
  18. Store particles at 4℃ in 3×ssc buffer

EDAC conjugation

  1. Pipet 12.5mg of polystirene-beads into a 1.5mL tube.
  2. Pellet the micro-beads via centrifugation for 5 minutes at 1000 xG.
  3. Resuspend micro-beads pellet in 0.4 ml of PolyLink Coupling Buffer.
  4. Pellet again via centrifugation for 5 minutes at 1000 G.
  5. Resuspend the micro-beads pellet in 0.17 ml of PolyLink Coupling Buffer.
  6. Just before use, prepare a 200 mg/ml EDAC solution by dissolving 10mg PolyLink EDAC in 50µl Polylink Coupling Buffer.
  7. Add 20 µl of the EDAC solution to the micro-beads suspension.
  8. Mix gently end-over-end or briefly vortex.
  9. Add 5 nmol of aminated DNA. Mix gently end-over-end or briefly vortex.
  10. Incubate for 90 minutes at room temperature with gentle mixing.
  11. Centrifuge mixture for 10 minutes at 1000 x G.
  12. Resuspend micro-beads pellet in 0.4ml Polylink Wash/Storage Buffer.
  13. Repeat Steps 12-13 for three times.
  14. Store particles at 4˚C in Polylink Wash/Storage Buffer.

PAGE protocol

  1. Make 20% polyacrylamide gel.
  2. Put into a microwave oven and liquefy urea for 15 seconds.
  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 becomes coagulate(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 + 1×TBE 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.

Observation of the platinum particles

  1. Make a solvent of about 100μL in an Eppendorf tube.
  2. Put beads in another Eppendorf tube with a spatula.
  3. Remove the dust on the surface of silicon rubber with the like Scotch tape.
  4. Drill holes in the silicon rubber with a belt punch.
  5. Press the holed silicon rubber to the surface of a dish.
  6. Add the beads in the tube to the solvent.
  7. Shake the tube by voltex and set in the Ultrasound device.
  8. Inject the beads solution into the hole of silicon rubber.
  9. Set the dish to the observation units after down the objective lens to the bottom.
  10. Observe with the eyepiece.

Azobenzen Protocol

Preparation

Solution A

solution amount strength
100μM seqAazo4_5thiol 5μL 10μM
2xSSC 25μL 1x
MlliQ water 20μL
Mass 50μL
  • 100μM seqAazo4_5thiol(DNAⅰ) 5μL
  • 2xSSC 25μL
  • MlliQ water 20μL

solution B

solution amount strength
100μM seqAc_5thiol 5μL 10μM
2xSSC 25μL 1x
MlliQ water 20μL
Mass 50μL
  • 100μM seqAc_5thiol(DNAⅱ) 5μl
  • 2xSSC 25μL
  • MlliQ water 20μL


Solution A+B

solution amount strength
A 30μL (DNAⅰ)5μM
B 30μL (DNAⅱ)5μM
Mass 60μL
  • solution A 30μL
  • solution B 30μL


Finally prepared solution

solution amount
Solution A 20μL
Solution B 20μL
Solution A+B 60μL


Way to Anneal DNA

  1. Irradiate visible-light to solutions A, B and A+B with Visible-light irradiation equipment(Epi-Green Slim pro S) to stabilize trans-azobanzen.
  2. Soaked solutions A, B, and A+B in a hot water of 100 digrees for 10 minute.
  3. Left these solutions A, B, and A+B for 50 minute, irradiating them visible-light.
  4. Put solutions into darkroom for 60 minute to cool down for stabilizing DNA duplex


Irradiate UV-light to DNA duplex and measures absorbance

  1. Irradiated UV-light(365nm) to a solution A+B for 1, 5, 30 minute.
  2. Measured absorbance of each DNA solutions(1~6) near 260nm wavelength of light. The DNA solutions we measured was as follows(table below).


No Solusion (time exposed UV-light)
1 A(0)
2 B(0)
3 A+B(0)
4 A+B(1)
5 A+B(5)
6 A+B(30)

Ascertain that DNA form duplex in 1~5% H₂O₂ solution without degenerated by H₂O₂

Way to make samples

20% polyacrilamide gels

solution amount strength
40%acrylamide gel 5mL 20%
10x TBE 1mL 1x
Milli-Q 4mL
Mass 10 mL


  • 40% acrylamide gel 5mL
  • 10x TBE 1mL
  • MilliQ 4mL


Way to make samples for electrophoresis

Make solutions ①~⑦ as table below

No.
solution amount strengthen amount strengthen amount strengthen
Milli-Q water 18µL 16µL 8µL
10%H₂O₂ 0µL 0% 2µL 1% 10µL 5%
100µM ssDNA F 0.5µL 2.5µM 0.5µL 2.5µM 0.5µL 2.5µM
100µM ssDNA R 0.5µL 2.5µM 0.5µL 2.5µM 0.5µL 2.5µM
10xSSC 1µL 0.5x 1µL 0.5x 1µL 0.5x
Mass 20 µL 20µL 20µL
No.
solution amount strengthen amount strengthen amount strengthen amount strengthen
Milli-Q water 18.5µL 8.5µL 18.5µL 8.5µL
10%H₂O₂ 0µL 0% 10µL 1% 0µL 5% 10
100µM ssDNA F 0.5µL 2.5µM 0.5µL 2.5µM 0µL 0µM 0µL 0µM
100µM ssDNA R 0µL 0µM 0µL 0µM 0.5µL 2.5µM 0.5µL 2.5µM
10xSSC 1µL 0.5x 1µL 0.5x 1µL 0.5x 1µL 0.5x
Mass 20μL 20μL 20μL 20μL
2. Incubate for 90minutes at room temperature with gentle mixing.
3.anealing complementary strands for 10 minutes at 80℃
4. Incubate for 90minutes at room temperature with gentle mixing.
5. Load wells with sample solution 5ul.(sample 3ul + 2x BPB solution 3ul = sample solution 6ul)
6. Run at specified voltage (250V or 300V) for specified time (50 minutes)
7. Put the gels into a cyber gold solution taper.(SYBR-Gold solution: 10000x cyber gold 20ul + MilliQ water 200ml)
8. Shake the taper by hand for 10 minutes.(using used cyber gold solution, shake for 15 or 20 minutes)
9. Take out the gels.
10. Spot blue light to observe the gel.
11. Take pictures through an orange filter by camera.

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
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
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
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
Suberic acid bis(3-sulfo-N-hydroxysuccinimide ester) sodium salt *** SIGMA S5799-25MG *** 051M4067V
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 (10X), 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



Laboratory instrument

  • Micro fine machining center
Roland MODELA MDX-40A
http://www.rolanddg.com/product/3d/3d/mdx-40a/index.html
  • Mili-Q water
http://www.millipore.com/catalogue/item/zrqsvp0ww
  • 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
LMS SHM2001
http://www.lms.co.jp/commodity/cat1/cat0101/post_43.html
  • 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
Hozan K-100
http://www.hozan.co.jp/E/catalog/Metal_working/K100.html
  • 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.
→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
Personal tools