(27 intermediate revisions by the same user not shown) | |||
Line 1: | Line 1: | ||
{{HokkaidoU_Japan}} | {{HokkaidoU_Japan}} | ||
{{Team:HokkaidoU_Japan/CSS}} | {{Team:HokkaidoU_Japan/CSS}} | ||
+ | {{HokkaidoU_Japan/footer}} | ||
Line 27: | Line 28: | ||
− | + | ||
<br> | <br> | ||
Line 41: | Line 42: | ||
<th>Solution</th> | <th>Solution</th> | ||
<td>template DNA</td> | <td>template DNA</td> | ||
− | <td>Primer-F 10µM</td> | + | <td>Primer-F 10 µM</td> |
− | <td>Primer-R 10µM</td> | + | <td>Primer-R 10 µM</td> |
<td>MgSO<sub>4</sub></td> | <td>MgSO<sub>4</sub></td> | ||
<td>dNTPs</td> | <td>dNTPs</td> | ||
Line 76: | Line 77: | ||
</tr> | </tr> | ||
<tr align="center"> | <tr align="center"> | ||
− | <td>3</td><td>68</td><td> | + | <td>3</td><td>68</td><td>30 / 1kbp</td> |
</tr> | </tr> | ||
<tr align="center"> | <tr align="center"> | ||
Line 82: | Line 83: | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | Cycle: | + | Cycle: sequence 2~3 × (25~45) |
<h2>3STEP Cycle (Tm value < 63°C)</h2> | <h2>3STEP Cycle (Tm value < 63°C)</h2> | ||
Line 99: | Line 100: | ||
</tr> | </tr> | ||
<tr align="center"> | <tr align="center"> | ||
− | <td>4</td><td>68</td><td> | + | <td>4</td><td>68</td><td>30 / 1kbp</td> |
</tr> | </tr> | ||
<tr align="center"> | <tr align="center"> | ||
Line 105: | Line 106: | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | Cycle: | + | Cycle: sequence 2~4 × (25~45) |
<h2>STEP DOWN</h2> | <h2>STEP DOWN</h2> | ||
Line 119: | Line 120: | ||
</tr> | </tr> | ||
<tr align="center"> | <tr align="center"> | ||
− | <td>3</td><td>74</td><td> | + | <td>3</td><td>74</td><td>30 / 1kbp</td> |
</tr> | </tr> | ||
<tr align="center"> | <tr align="center"> | ||
Line 148: | Line 149: | ||
</table> | </table> | ||
Cycle: | Cycle: | ||
− | <br> | + | <br>sequence 2~3 × 5 |
− | <br> | + | <br>sequence 4~5 × 5 |
− | <br> | + | <br>sequence 6~7 × 5 |
− | <br> | + | <br>sequence 8~9 × 15 |
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp1").style.display="none"; | document.getElementById("disp1").style.display="none"; | ||
Line 185: | Line 187: | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp2").style.display="none"; | document.getElementById("disp2").style.display="none"; | ||
Line 211: | Line 214: | ||
<th>Solution</th> | <th>Solution</th> | ||
<td>DNA</td> | <td>DNA</td> | ||
− | <td>RE1 | + | <td>RE1 10 U/µL</td> |
− | <td>RE2 | + | <td>RE2 10 U/µL</td> |
<td>Appropriate buffer</td> | <td>Appropriate buffer</td> | ||
<td>Total</td> | <td>Total</td> | ||
Line 264: | Line 267: | ||
<div id="disp4"> | <div id="disp4"> | ||
<span class="nomal2"> | <span class="nomal2"> | ||
− | Mix the following reagents in 0.2 mL PCR tube. Use DNA Ligation Kit | + | Mix the following reagents in 0.2 mL PCR tube. Use DNA Ligation Kit Mighty Mix (Takara Bio Inc.) which contains ligase and buffer. |
<table> | <table> | ||
Line 291: | Line 294: | ||
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp4").style.display="none"; | document.getElementById("disp4").style.display="none"; | ||
Line 314: | Line 318: | ||
<ol> | <ol> | ||
<li>Put gel into electrophoresis tank.</li> | <li>Put gel into electrophoresis tank.</li> | ||
− | <li> | + | <li>Pour 2x TBE buffer into the tank to soak gel.</li> |
− | <li>Add 5 µL of EtBr into | + | <li>Add 5 µL of EtBr into cathode.</li> |
<li>Pre-migration for 30 min at 100 V.</li> | <li>Pre-migration for 30 min at 100 V.</li> | ||
− | <li>Apply DNA | + | <li>Apply DNA solutions with 6x loading dye and ladder.</li> |
<li>Start electrophoresis at 100 V.</li> | <li>Start electrophoresis at 100 V.</li> | ||
+ | <li>Stop at appropriate time. | ||
</ol> | </ol> | ||
</span> | </span> | ||
− | |||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp5").style.display="none"; | document.getElementById("disp5").style.display="none"; | ||
Line 350: | Line 355: | ||
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp6").style.display="none"; | document.getElementById("disp6").style.display="none"; | ||
Line 378: | Line 384: | ||
<li>Remove supernatant and add 600 µL of 70% ethanol.</li> | <li>Remove supernatant and add 600 µL of 70% ethanol.</li> | ||
<li>Centrifuge at 15,000 rpm for 5 min at 25°C.</li> | <li>Centrifuge at 15,000 rpm for 5 min at 25°C.</li> | ||
− | <li>Remove supernatant and | + | <li>Remove supernatant and dry up at room temperature with light sheilding.</li> |
<li>Suspend with 10 µL of TE.</li> | <li>Suspend with 10 µL of TE.</li> | ||
</ol> | </ol> | ||
− | |||
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp7").style.display="none"; | document.getElementById("disp7").style.display="none"; | ||
Line 411: | Line 417: | ||
<th>Solution</th> | <th>Solution</th> | ||
<td>DNA</td> | <td>DNA</td> | ||
− | <td>Kapa-Taq ( | + | <td>Kapa-Taq (Taq polymerase)</td> |
− | <td>EX-F primer 10µM</td> | + | <td>EX-F primer 10 µM</td> |
− | <td>PS-R primer 10µM</td> | + | <td>PS-R primer 10 µM</td> |
<td>Total</td> | <td>Total</td> | ||
</tr> | </tr> | ||
Line 437: | Line 443: | ||
</tr> | </tr> | ||
<tr align="center"> | <tr align="center"> | ||
− | <td>3</td><td>68</td><td>60 | + | <td>3</td><td>68</td><td>60 / 1kbp</td> |
</tr> | </tr> | ||
<tr align="center"> | <tr align="center"> | ||
Line 443: | Line 449: | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | Cycles: | + | Cycles: sequence 2~3 × 25~45 |
− | + | ||
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp8").style.display="none"; | document.getElementById("disp8").style.display="none"; | ||
Line 472: | Line 478: | ||
<th>Solution</th> | <th>Solution</th> | ||
<td>5 x Sequencing Buffer</td> | <td>5 x Sequencing Buffer</td> | ||
− | <td>primer 1µM</td> | + | <td>primer 1 µM</td> |
<td>template DNA</td> | <td>template DNA</td> | ||
<td>Ready Reaction Premix</td> | <td>Ready Reaction Premix</td> | ||
Line 506: | Line 512: | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | Cycle: | + | Cycle: sequence 2~4 × 25 |
Line 529: | Line 535: | ||
</table> | </table> | ||
<ol> | <ol> | ||
− | <li>Centrifuge at 15,000 rpm for 15 min at room temprature</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>Centrifuge at 15,000 rpm for 10 min at room temprature</li> | + | <li>Centrifuge at 15,000 rpm for 10 min at room temprature.</li> |
− | <li>Remove supernatant and | + | <li>Remove supernatant and dry up 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 538: | Line 544: | ||
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp9").style.display="none"; | document.getElementById("disp9").style.display="none"; | ||
Line 566: | Line 573: | ||
<li>Add 5 µL, 50 µL, and 500 µL of original cells to 100 mL of LB.</li> | <li>Add 5 µL, 50 µL, and 500 µL of original cells to 100 mL of LB.</li> | ||
<li>Incubate the cells at 130 rpm at 20°C, until OD<sub>600</sub> reach 0.5.</li> | <li>Incubate the cells at 130 rpm at 20°C, until OD<sub>600</sub> reach 0.5.</li> | ||
− | <li>Take 50 mL of incubated cells to two | + | <li>Take 50 mL of incubated cells to two different culture tubes and centrifuge them at 3,000 rpm for 20 min at 4°C.</li> |
<li>Remove supernatant and add 75 mL of TB to each tube.</li> | <li>Remove supernatant and add 75 mL of TB to each tube.</li> | ||
<li>Bring them to a one tube and centrifuge at 3,000 rpm for 20 min at 4°C.</li> | <li>Bring them to a one tube and centrifuge at 3,000 rpm for 20 min at 4°C.</li> | ||
Line 575: | Line 582: | ||
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp10").style.display="none"; | document.getElementById("disp10").style.display="none"; | ||
Line 600: | Line 608: | ||
<li>Incubate on ice for 30 min.</li> | <li>Incubate on ice for 30 min.</li> | ||
<li>Add to LB.</li> | <li>Add to LB.</li> | ||
− | <li> | + | <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 37°C for 16~20 hrs.</li> | <li>Incubate the plate(s) at 37°C for 16~20 hrs.</li> | ||
Line 606: | Line 614: | ||
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp11").style.display="none"; | document.getElementById("disp11").style.display="none"; | ||
Line 631: | Line 640: | ||
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp12").style.display="none"; | document.getElementById("disp12").style.display="none"; | ||
Line 655: | Line 665: | ||
<li>Pick the colony with an inoculating loop from the agar plate.</li> | <li>Pick the colony with an inoculating loop from the agar plate.</li> | ||
<li>Drag the loop across on a new agar plate.</li> | <li>Drag the loop across on a new agar plate.</li> | ||
− | <li> | + | <li>Resterilize the loop and drag it across again.</li> |
<li>Repeat method 3.</li> | <li>Repeat method 3.</li> | ||
</ol> | </ol> | ||
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp13").style.display="none"; | document.getElementById("disp13").style.display="none"; | ||
Line 687: | Line 698: | ||
<li>Remove supernatant and add 100 µL of 70% ethanol.</li> | <li>Remove supernatant and add 100 µL of 70% ethanol.</li> | ||
<li>Centrifuge at 15,000 rpm for 2 min at 4°C.</li> | <li>Centrifuge at 15,000 rpm for 2 min at 4°C.</li> | ||
− | <li>Remove supernatant and | + | <li>Remove supernatant and dry up at room temperature with light sheilding.</li> |
<li>Suspend with 10 µL of TE.</li> | <li>Suspend with 10 µL of TE.</li> | ||
</ol> | </ol> | ||
</span> | </span> | ||
</div> | </div> | ||
+ | |||
<script> | <script> | ||
document.getElementById("disp14").style.display="none"; | document.getElementById("disp14").style.display="none"; | ||
Line 720: | Line 732: | ||
<th>Solution</th> | <th>Solution</th> | ||
<td>template DNA</td> | <td>template DNA</td> | ||
− | <td>5'-biotinylated 100-UP primer 10µM</td> | + | <td>5'-biotinylated 100-UP primer 10 µM</td> |
− | <td>5'-biotinylated 200-DN primer 10µM</td> | + | <td>5'-biotinylated 200-DN primer 10 µM</td> |
<td>MgSO<sub>4</sub></td> | <td>MgSO<sub>4</sub></td> | ||
<td>dNTPs</td> | <td>dNTPs</td> | ||
Line 755: | Line 767: | ||
</tr> | </tr> | ||
<tr align="center"> | <tr align="center"> | ||
− | <td>3</td><td>68</td><td> | + | <td>3</td><td>68</td><td>30 / 1kbp</td> |
</tr> | </tr> | ||
<tr align="center"> | <tr align="center"> | ||
Line 761: | Line 773: | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | Cycle: | + | Cycle: sequence 2~3 × (25~45) |
<h2>Preparation of magnetic beads</h2> | <h2>Preparation of magnetic beads</h2> | ||
Line 789: | Line 801: | ||
</span> | </span> | ||
</div> | </div> | ||
− | < | + | |
+ | <br> | ||
+ | <br> | ||
<script> | <script> | ||
document.getElementById("disp15").style.display="none"; | document.getElementById("disp15").style.display="none"; | ||
Line 810: | Line 824: | ||
+ | </div> | ||
+ | <!-- 2016contents 閉じる --> | ||
+ | |||
+ | <!--begin footer--> | ||
+ | <br> | ||
+ | <div id="footer" style="background-color: #97D3E3; position:relative;width:100%";> | ||
+ | <div id="footer-logo"> | ||
+ | <br> | ||
+ | <br> | ||
+ | |||
+ | <a href="https://www.facebook.com/igem.hokkaido.u.bio"> | ||
+ | <img style="height:50px;position:relative;" src="https://static.igem.org/mediawiki/2016/9/96/T--HokkaidoU_Japan--facebook.png" alt="Facebook"></a> | ||
+ | |||
+ | <a href="https://twitter.com/igem_hokkaidou"> | ||
+ | <img style="height:50px;position:relative;" src="https://static.igem.org/mediawiki/2016/1/13/T--HokkaidoU_Japan--twitter.png" alt="Twitter"></a> | ||
+ | |||
+ | <a href="mailto:igemhokkaidou@gmail.com"> | ||
+ | <img style="height:50px;position:relative;" src="https://static.igem.org/mediawiki/2016/4/4e/T--HokkaidoU_Japan--mail.png" alt="E-mail"></a> | ||
+ | |||
+ | <a href="http://igemhokkaidou.wordpress.com"> | ||
+ | <img style="height:50px;position:relative;" src="https://static.igem.org/mediawiki/2016/3/3b/T--HokkaidoU_Japan--icon1.png" alt="Web"> | ||
+ | </a> | ||
+ | |||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <!--background--> | ||
</body> | </body> | ||
</html> | </html> |
Latest revision as of 19:25, 19 October 2016
Team:HokkaidoU Japan
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
Cycles: sequence 2~3 × 25~45
Cycle: sequence 2~4 × 25
Thermal protocol is following
Cycle: sequence 2~3 × (25~45)
![Protcols](https://static.igem.org/mediawiki/2016/2/29/T--HokkaidoU_Japan--protocols.png)
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.
Thermal protocol is following
Cycle: sequence 2~3 × (25~45)
Cycle: sequence 2~4 × (25~45)
Cycle:
sequence 2~3 × 5
sequence 4~5 × 5
sequence 6~7 × 5
sequence 8~9 × 15
Solution | template DNA | Primer-F 10 µM | Primer-R 10 µM | MgSO4 | dNTPs | 10x Buffer | KOD Plus Neo | DW | Total |
---|---|---|---|---|---|---|---|---|---|
Volume (µL) | 1 | 1 | 1 | 3 | 5 | 5 | 1 | 33 | 50 |
2STEP Cycle (Tm value > 63°C)
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 98 | 10 |
3 | 68 | 30 / 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 | 30 / 1kbp |
5 | 4 | Hold |
STEP DOWN
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 98 | 10 |
3 | 74 | 30 / 1kbp |
4 | 98 | 10 |
5 | 72 | 30 |
6 | 98 | 10 |
7 | 70 | 30 |
8 | 98 | 10 |
9 | 68 | 30 |
10 | 68 | 420 |
11 | 4 | Hold |
sequence 2~3 × 5
sequence 4~5 × 5
sequence 6~7 × 5
sequence 8~9 × 15
PCR Purification
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
Purification of PCR products
Purification of PCR products
Digestion
Mix the following reagents in PCR tube.
Solution | DNA | RE1 10 U/µL | RE2 10 U/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 (Takara Bio Inc.) which contains ligase and buffer.
Thermal protocol is following
Solution | Vector DNA | Insert DNA | DW | Mighty Mix | Total |
---|---|---|---|---|---|
Volume (µL) | 1 | 2 | 2 | 5 | 10 |
Sequence | Temp. (°C) | Time (min) |
---|---|---|
1 | 16 | 30 |
2 | 65 | 10 |
3 | 4 | Hold |
Electrophoresis
- Put gel into electrophoresis tank.
- Pour 2x TBE buffer into the tank to soak gel.
- Add 5 µL of EtBr into cathode.
- Pre-migration for 30 min at 100 V.
- Apply DNA solutions with 6x loading dye and ladder.
- Start electrophoresis at 100 V.
- Stop at appropriate time.
Gel Extraction
FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co., Ltd)
DNA extraction from gel
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 dry up at room temperature with light sheilding.
- Suspend with 10 µL of TE.
Colony PCR
Solution | DNA | Kapa-Taq (Taq polymerase) | EX-F primer 10 µM | PS-R primer 10 µM | 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 / 1kbp |
4 | 4 | Hold |
Sequencing
Solution | 5 x Sequencing Buffer | primer 1 µM | 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 dry up 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 different 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
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.
- Resterilize 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 dry up 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 10 µM | 5'-biotinylated 200-DN primer 10 µM | MgSO4 | dNTPs | 10x Buffer | KOD Plus Neo | DW | Total |
---|---|---|---|---|---|---|---|---|---|
Volume (µL) | 1 | 1.5 | 1.5 | 3 | 5 | 5 | 1 | 32 | 50 |
2STEP Cycle (Tm value > 63°C)
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 98 | 10 |
3 | 68 | 30 / 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.