Biomod/2011/TeamJapan/Tokyo/Notebook/Protocols: Difference between revisions
Line 102: | Line 102: | ||
==DNA attaching to grass beads protocol== | ==DNA attaching to grass beads protocol== | ||
This protocol is not used for DNA ciliate project because | '''This protocol is not used for DNA ciliate project because''' | ||
*In this protocol, the phrase “displace A (200 ul) with B (200 ul)” comes repeatedly. This mean is the following. | *In this protocol, the phrase “displace A (200 ul) with B (200 ul)” comes repeatedly. This mean is the following. (“the tube” is poured A (200 ul) at first)<br / >Mix the tube by inverse agitation<br / >The tube is centrifuged by the Chibitan (personal centrifuge) for 30 seconds<br / >Remove the supernatant of the tube<br / >Pour B (200 ul) into the tube.<br / >(The mean of “displace A (200 ul) with B (200 ul) four times” is operating this sequential process repeats four times.) | ||
(“the tube” is poured A (200 ul) at first) | |||
Mix the tube by inverse agitation | |||
#Put glass beads 5mg into a 0.6ml tube | #Put glass beads 5mg into a 0.6ml tube | ||
#Pour 1M NaOH 200 ul into the tube | #Pour 1M NaOH 200 ul into the tube |
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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 Zn(2+).)
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℃) for 1 minute to separate substrate DNA from deoxyribozime DNA
- Centrifuge (15000rpm,1minute) to make precipitation
- Put the tube onto heating block (80℃) 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 1um polystyrene beads
- Put 200ul of 2.5% polystyrene beads solution^(1)) into a 1.5ml tube.
- Centrifuge the tube for 1minute at 15,000rpm, and then remove the supernatant.
- Put 200ul PBS buffer (pH 8.0) into the tube, and disperse the beads by pipetting or vortex mixer.
- Centrifuge the tube for 1minute at 15,000rpm, and then remove the supernatant.
- Repeat 3, 4 for three times to displace the solvent to PBS buffer.
- Put 800ul PBS buffer (pH 8.0) into the tube, and disperse the beads.
- Put 200ul of 1M NHS solution^(2)) into the tube, and mix with vortex mixer.
- Put 0.0384g EDC-HCl into the tube, and mix with vortex mixer to dissolve.
- React beads with NHS and EDC-HCl solution using vortex mixer for an hour.
- Centrifuge the tube for 1minute at 15,000rpm, and then remove the supernatant.
- Put 200ul PBS buffer (pH 8.0) into the tube and disperse the beads by pipetting or vortex mixer.
- Centrifuge the tube for 1minute at 15,000rpm, and then remove the supernatant.
- Repeat 11, 12 for three times^(3)) to displace the solvent to PBS buffer.
- Put 8ul of 50uM DNA solution^(4)) into the tube, and mix by pipetting.
- React beads with DNA solution using the micro tube rotator for more than 2 hours.
- Centrifuge the tube for 1minute at 15,000rpm, and then remove the supernatant.
- Put 200ul of 0.2% SDS solution into the tube, and mix by pipetting and vortex mixer.
- Centrifuge the tube for 1minute at 15,000rpm, and then remove the supernatant.
- Put 200ul of 0.1M Tris-HCl buffer into the tube, and disperse the beads by pipetting or vortex mixer.
- Centrifuge the tube for 1minute at 15,000rpm, and then remove the supernatant.
- Repeat 19, 20 for three times^(3)) to displace the solvent to 0.1M Tris-HCl buffer.
- Put 200ul of 0.1M Tris-HCl buffer into the tube, and mix with vortex mixer.
- React beads with Tris-HCl buffer using the micro tube rotator for an hour.
- Keep the tube in refrigerator (4 deg C)
Notes
- The density of polystyrene beads is about 1.1g/ml, so 200ul of 2.5% polystyrene beads solution contains about 5mg polystyrene beads.
- Prepare 1M NHS solution by dissolving 0.023g NHS in 200ul PBS buffer (pH 8.0)
- This process should be done quickly because NHS can be easily hydrolyzed.
DNA attaching to grass beads protocol
This protocol is not used for DNA ciliate project because
- In this protocol, the phrase “displace A (200 ul) with B (200 ul)” comes repeatedly. This mean is the following. (“the tube” is poured A (200 ul) at first)
Mix the tube by inverse agitation
The tube is centrifuged by the Chibitan (personal centrifuge) for 30 seconds
Remove the supernatant of the tube
Pour B (200 ul) into the tube.
(The mean of “displace A (200 ul) with B (200 ul) four times” is operating this sequential process repeats four times.)
- Put glass beads 5mg into a 0.6ml tube
- Pour 1M NaOH 200 ul into the tube
- Wash glass beads with NaOH using the micro tube rotator for an hour
- Displacing NaOH (200 ul) with MilliQ water (200 ul) four times
- Displacing MilliQ water (200 ul) with silane solution (200 ul) four times
- React glass beads react with silane solution using the micro tube rotator for an hour
- Displacing silane solution (200 ul) with MilliQ water (200 ul) in three times
- Displacing MilliQ water (200 ul) with DSS solution (200 ul) four times
- React glass beads with DSS solution using the micro tube rotator an hour
- Displacing DSS solution (200 ul) with 1x PBS(-) (200 ul ) three times
- Mix the tube by inverse agitation
- Be centrifuged by the Chibitan (personal centrifuge)
- Remove the supernatant of the tube
- Pour 100 uM DNA solution (4ul) and 2x PBS(-) (4 ul) into the tube
- Add 2x PBS(-) to the solution until glass beads is flooded
- React glass beads with DNA using the micro tube rotator overnight
- Displace DNA solution with SDS solution (100 ul) three times
- Displace SDS solution (100 ul) with Tris-HCl (200 ul) four times
- React glass beads with Tris-HCl using the micro tube rotator for a half hour
- Displace Tris-HCl (200 ul) with 3% BSA solution (200 ul) four times
- Do BSA blocking using the micro tube rotator for a half hour
- Displace 3% BSA solution (200 ul) with Tris-HCl (200 ul) four times
- Keep the tube in refrigerator (4 deg C)
- Micro tube rotator… MTR-103 (AS ONE)
UV switch protocol
- UV switch protocol (To check by electrophoresis)
- A…UV-switching-trap-DNA
- 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 5x SSC 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 Mg(2+) in 5x SSC solution)
(To make controls, don’t mix all solutions) - Open the tube’s caps and spot visible light (no UV) to the solutions for 30 min.The distance from solutions to light is 9 cm.
- Close the tube’s caps.
- Turn on the heating blocks at 95℃.
- Put tubes onto heating block for 2 min. (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 hr.
- 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. (link)
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, Mg(2+) 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(2+) in 5x SSC solution is used for dilutions