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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
How to make DNA ciliate by EDAC
We used "PolyLink Protein Coupling Kit for COOH Microspheres" in this protocol.
Pipet 12.5mg of micro-beads into a 1.5mL tube.
Pellet the micro-beads via centrifugation for 5-10 minutes at approximately 500-1000 xG.
Resuspend micro-beads pellet in 0.4ml of Polylink Coupling Buffer.
Pellet again via centrifugation for 5-10 minutes at approximately 500-1000 G.
Resuspend the micro-beads pellet in 0.17ml of Polylink Coupling Buffer.
Just before use, prepare a 200mg/ml EDAC solution by dissolving 10mg Polylink EDAC in 50µl Polylink Coupling Buffer.
Add 20µl of the EDAC solution to the micro-beads suspension.
Mix gently end-over-end or briefly vortex.
Add 5nmol of aminated DNA. Mix gently end-over-end or briefly vortex.
Incubate for 30-60 minutes at room temperature with gentle mixing.
Centrifuge mixture for 10 minutes at approximately 500-1000 x G.
Resuspend micro-beads pellet in 0.4ml Polylink Wash/Storage Buffer.
Repeat Steps 12-13.
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
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
Immobilization of single-stranded DNA on glass plate using microchannel
Place a MAS coated glass plate in 5mM DSS solution for an hour.
Wash thrice the glass plate in Milli-Q water, and dry it completely.
PDMS mold to overlay on the glass plate.
Use a micropippet to add 20uM DNA solution droplets to inlets of the microchannel.
Satisfying solution in the microchannels by the passive pumping.
Incubate for 2 hours at room temperature.
Wash the glass plate in 0.2% SDS solution.
Wash thrice the glass plate in Mili-Q water, and dry it completely.
Place the glass plate in 3% BSA solution for 30 minutes.
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
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.
