Line 32: | Line 32: | ||
</div> | </div> | ||
− | |||
<h1>Protocols</h1> | <h1>Protocols</h1> | ||
Line 69: | Line 68: | ||
</table> | </table> | ||
<p>Thermal protocol is following</p> | <p>Thermal protocol is following</p> | ||
− | <h3>2STEP Cycle (Tm value | + | <h3>2STEP Cycle (Tm value > 63°C)</h3> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 89: | Line 88: | ||
Cycle: sequence2~3 × (25~45) | Cycle: sequence2~3 × (25~45) | ||
− | <h3>3STEP Cycle (Tm value | + | <h3>3STEP Cycle (Tm value < 63°C)</h3> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 256: | Line 255: | ||
<th>Solution</th> | <th>Solution</th> | ||
<td>5 x Sequencing Buffer</td> | <td>5 x Sequencing Buffer</td> | ||
− | <td>primer 1µ | + | <td>primer 1µM</td> |
<td>template DNA</td> | <td>template DNA</td> | ||
<td>Ready Reaction Premix</td> | <td>Ready Reaction Premix</td> | ||
Line 313: | Line 312: | ||
</table> | </table> | ||
<ol> | <ol> | ||
− | <li> | + | <li>Centrifuge at 15,000 rpm for 15 min at room temprature</li> |
<li>Remove supernatant ,add 100 µL of 70% EtOH and tap tubes by finger.</li> | <li>Remove supernatant ,add 100 µL of 70% EtOH and tap tubes by finger.</li> | ||
− | <li> | + | <li>Centrifuge at 15,000 rpm for 10 min at room temprature</li> |
− | <li>Remove supernatant and air dry at room temperature | + | <li>Remove supernatant and air dry at room temperature.</li> |
− | + | ||
<li>Resuspend the pellet to HiDi formamide and remove to 96-well plate.</li> | <li>Resuspend the pellet to HiDi formamide and remove to 96-well plate.</li> | ||
<li>Set the plate and start electrophoresis.</li> | <li>Set the plate and start electrophoresis.</li> | ||
Line 343: | Line 341: | ||
<li>Add plasmid to thawed competent cells on ice.</li> | <li>Add plasmid to thawed competent cells on ice.</li> | ||
<li>Incubate on ice for 30 min.</li> | <li>Incubate on ice for 30 min.</li> | ||
− | <li>Add LB.</li> | + | <li>Add to LB.</li> |
− | <li>(Incubate the cells for 2 hrs at | + | <li>(Incubate the cells for 2 hrs at 37°C.)</li> |
<li>Spread 300 µL of the culture onto plate with LB and appropriate antibiotics.</li> | <li>Spread 300 µL of the culture onto plate with LB and appropriate antibiotics.</li> | ||
− | <li>Incubate the plate(s) at | + | <li>Incubate the plate(s) at 37°C for 16~20 hrs.</li> |
</ol> | </ol> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
Line 397: | Line 383: | ||
<th>Solution</th> | <th>Solution</th> | ||
<td>template DNA</td> | <td>template DNA</td> | ||
− | <td>5'-biotinylated | + | <td>5'-biotinylated 100-UP primer 10µM</td> |
− | <td>5'-biotinylated | + | <td>5'-biotinylated 200-DN primer 10µM</td> |
<td>MgSO<sub>4</sub></td> | <td>MgSO<sub>4</sub></td> | ||
<td>dNTPs</td> | <td>dNTPs</td> | ||
Line 420: | Line 406: | ||
</table> | </table> | ||
<p>Thermal protocol is following</p> | <p>Thermal protocol is following</p> | ||
− | <h3>2STEP Cycle (Tm value | + | <h3>2STEP Cycle (Tm value > 63°C)</h3> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 443: | Line 429: | ||
<ol> | <ol> | ||
<li>Mix 2 µL of magnetic beads (SiMAG-Streptavidin) and 48 µL of TE by vibration using sonic-toothbrush.</li> | <li>Mix 2 µL of magnetic beads (SiMAG-Streptavidin) and 48 µL of TE by vibration using sonic-toothbrush.</li> | ||
− | <li>Collect the beads by attracting them to one side in 0.2 mL polypropylene tube using neodymium magnet | + | <li>Collect the beads by attracting them to one side in 0.2 mL polypropylene tube using neodymium magnet.</li> |
<li>Remove supernatant.<li> | <li>Remove supernatant.<li> | ||
</ol> | </ol> | ||
Line 457: | Line 443: | ||
<h3>Double restriction digestion<h3> | <h3>Double restriction digestion<h3> | ||
<ol> | <ol> | ||
− | <li>Add Digestion Premix containing 1 µL of 10x RE solution, 8 µL of DW and each 0.5 µL of restriction endonuclease to the beads.</li> | + | <li>Add Digestion Premix containing 1 µL of 10x RE solution, 8 µL of DW and each 0.5 µL of restriction endonuclease, <I>Xba</I>I and <I>Spe</I>I, to the beads.</li> |
<li>Mix by pumping using pipette.</li> | <li>Mix by pumping using pipette.</li> | ||
<li>Incubate at 37 °C for 30 min.</li> | <li>Incubate at 37 °C for 30 min.</li> |
Revision as of 03:04, 17 August 2016
Team:HokkaidoU Japan
Document the dates you worked on your project.
What should this page have?
- Chronological notes of what your team is doing.
- Brief descriptions of daily important events.
- Pictures of your progress.
- Mention who participated in what task.
Inspiration
You can see what others teams have done to organize their notes:
Protocols
PCR
Use KOD -Plus- Neo (TOYOBO CO.,LTD) as polymerase. Mix PCR solutions and run the PCR machine in a program which is detailed below.
Solution | template DNA | Primer-F 10µM | Primer-R 10µM | MgSO4 | dNTPs | 10x Buffer | KOD Plus Neo | DW | Total |
---|---|---|---|---|---|---|---|---|---|
Volume (µL) | 1 | 1 | 1 | 3 | 5 | 5 | 1 | 33 | 50 |
Thermal protocol is following
2STEP Cycle (Tm value > 63°C)
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 98 | 10 |
3 | 68 | 30sec / 1kbp |
4 | 4 | Hold |
3STEP Cycle (Tm value < 63°C)
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 98 | 10 |
3 | Tm | 30 |
4 | 68 | 30sec / 1kbp |
5 | 4 | Hold |
PCR Purification
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
Purification of PCR products
Digestion
Mix the following reagents in PCR tube.Solution | DNA | RE1 10U/µL | RE2 10U/µL | Appropriate buffer | Total |
---|---|---|---|---|---|
Volume (µL) | 16 | 1 | 1 | 2 | 20 |
Sequence | Temp. (°C) | Time (min) |
---|---|---|
1 | 37 | 120 |
2 | 65 | 15 |
3 | 4 | Hold |
Ligation
Mix the following reagents in 0.2 mL PCR tube. Use DNA Ligation Kit <Mighty Mix&rt; (Takara Bio Inc.) which contains ligase and buffer.
Solution | Vector DNA | Insert DNA | DW | Mighty Mix | Total |
---|---|---|---|---|---|
Volume (µL) | 1 | 2 | 2 | 5 | 10 |
Thermal protocol is following
Sequence | Temp. (°C) | Time (min) |
---|---|---|
1 | 16 | 30 |
2 | 65 | 10 |
3 | 4 | Hold |
Electrophoresis
- Put gel into electrophoresis tank.
- Pore 2x TBE buffer into the tank to soak gel.
- Add 5 µL of EtBr into cathod.
- Pre-migration for 30 min at 100 V.
- Apply DNA solution with 6x loading dye and ladder.
- Start electrophoresis at 100 V.
Gel Extraction
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
DNA extraction from gel
Ethanol precipitation
- Add 1/10 volume of NaOAc, and 5/2 of 100% ethanol.
- Leave it at room temperature for 5 min.
- Centrifuge at 15,000 rpm for 15 min at 25°C.
- Remove supernatant and add 600 µL of 70% ethanol.
- Centrifuge at 15,000 rpm for 5 min at 25°C.
- Remove supernatant and air-dry at room temperature with light sheilding.
- Suspend with 10 µL of TE.
Colony PCR
Solution | DNA | Kapa-Taq (Taq polymerase) | EX-F primer 10µM | PS-R primer 10µM | Total |
---|---|---|---|---|---|
Volume (µL) | 4.2 | 5 | 0.4 | 0.4 | 10 |
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 94 | 30 |
3 | 68 | 60 sec / 1kbp | 4 | 4 | Hold |
Sequencing
Solution | 5 x Sequencing Buffer | primer 1µM | template DNA | Ready Reaction Premix | DW | Total |
---|---|---|---|---|---|---|
Volume (µL) | 1.5 | 1.5 | 1 | 1 | 5 | 10 |
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 96 | 10 |
2 | 50 | 5 |
3 | 60 | 240 |
4 | 4 | Hold |
Ethanol precipitation
Solution | PCR product | DW | 3M NaOAc | Glycogen | 100% EtOH |
---|---|---|---|---|---|
Volume (µL) | 10 | 10 | 2 | 1 | 50 |
- Centrifuge at 15,000 rpm for 15 min at room temprature
- Remove supernatant ,add 100 µL of 70% EtOH and tap tubes by finger.
- Centrifuge at 15,000 rpm for 10 min at room temprature
- Remove supernatant and air dry at room temperature.
- Resuspend the pellet to HiDi formamide and remove to 96-well plate.
- Set the plate and start electrophoresis.
Competent Cells
- Thaw original competent cells on ice.
- Add 5 µL of original competent cells to 2 mL of LB.
- Incubate the cells for 16 hrs at 37°C.
- Add 5 µL, 50 µL, and 500 µL of original cells to 100 mL of LB.
- Incubate the cells at 130 rpm at 20°C, until OD600 reach 0.5.
- Take 50 mL of incubated cells to two differnt culture tubes and centrifuge them at 3,000 rpm for 20 min at 4°C.
- Remove supernatant and add 75 mL of TB to each tube.
- Bring them to a one tube and centrifuge at 3,000 rpm for 20 min at 4°C.
- Remove supernatant and add 32 mL of TB.
- Add 32 µL of DMSO 10 times.
- Take 50 µL and freeze with liquid nitrogen.
Transformation
- Add plasmid to thawed competent cells on ice.
- Incubate on ice for 30 min.
- Add to LB.
- (Incubate the cells for 2 hrs at 37°C.)
- Spread 300 µL of the culture onto plate with LB and appropriate antibiotics.
- Incubate the plate(s) at 37°C for 16~20 hrs.
Mini-prep
FastGeneTM Plasmid mini kit (Nippon Genetics Co., Ltd)
fast / standard / low copy protocol
Streaking (Single colony isolation)
- Pick the colony with an inoculating loop from the agar plate.
- Drag the loop across on a new agar plate.
- Re-sterilise the loop and drag it across again.
- Repeat method 3.
PEG precipitation
- Add 13 µL of PEG to 20 µL of product(s).
- Leave it at room temperature for 1 hr.
- Centrifuge at 15,000 rpm for 20 min at 4°C.
- Remove supernatant and add 100 µL of 70% ethanol.
- Centrifuge at 15,000 rpm for 2 min at 4°C.
- Remove supernatant and air-dry at room temperature with light sheilding.
- Suspend with 10 µL of TE.
Gel Free System
Preparation of biotinylated DNA fragments
Use KOD -Plus- Neo (TOYOBO CO.,LTD) as polymerase and 5'-biotinylated primers. Mix PCR solutions and run the PCR machine in a program which is detailed below.
Solution | template DNA | 5'-biotinylated 100-UP primer 10µM | 5'-biotinylated 200-DN primer 10µM | MgSO4 | dNTPs | 10x Buffer | KOD Plus Neo | DW | Total |
---|---|---|---|---|---|---|---|---|---|
Volume (µL) | 1 | 1.5 | 1.5 | 3 | 5 | 5 | 1 | 32 | 50 |
Thermal protocol is following
2STEP Cycle (Tm value > 63°C)
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 98 | 10 |
3 | 68 | 30sec / 1kbp |
4 | 4 | Hold |
Preparation of magnetic beads
- Mix 2 µL of magnetic beads (SiMAG-Streptavidin) and 48 µL of TE by vibration using sonic-toothbrush.
- Collect the beads by attracting them to one side in 0.2 mL polypropylene tube using neodymium magnet.
- Remove supernatant.
Fixation to magnetic beads
- Add 3 µL of PCR product (0.48 pmol) and 7 µL of TE to beads.
- Mix by vibration using sonic-toothbrush.
- Collect the beads using magnet.
- Remove supernatant containing excess amount of free DNA fragment.
Double restriction digestion
- Add Digestion Premix containing 1 µL of 10x RE solution, 8 µL of DW and each 0.5 µL of restriction endonuclease, XbaI and SpeI, to the beads.
- Mix by pumping using pipette.
- Incubate at 37 °C for 30 min.
- Collect the beads using magnet.
- Obtain supernatant containing digested DNA fragment.
- Purify the supernatant by ethanol precipitation.
- Add Digestion Premix containing 1 µL of 10x RE solution, 8 µL of DW and each 0.5 µL of restriction endonuclease, XbaI and SpeI, to the beads.
- Mix by pumping using pipette.
- Incubate at 37 °C for 30 min.
- Collect the beads using magnet.
- Obtain supernatant containing digested DNA fragment.
- Purify the supernatant by ethanol precipitation.