(Prototype team page) |
|||
Line 32: | Line 32: | ||
</div> | </div> | ||
+ | |||
+ | |||
+ | <h1>Protocols</h1> | ||
+ | |||
+ | |||
+ | |||
+ | <h2>PCR</h2> | ||
+ | <p> | ||
+ | Use KOD -Plus- Neo (TOYOBO CO.,LTD) as polymerase. Mix PCR solutions and run the PCR machine in a program which is detailed below. | ||
+ | </p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Solution</th> | ||
+ | <td>template DNA</td> | ||
+ | <td>Primer-F 10µM</td> | ||
+ | <td>Primer-R 10µM</td> | ||
+ | <td>MgSO<sub>4</sub></td> | ||
+ | <td>dNTPs</td> | ||
+ | <td>10x Buffer</td> | ||
+ | <td>KOD Plus Neo</td> | ||
+ | <td>DW</td> | ||
+ | <td>Total</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Volume (µL)</th> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>3</td> | ||
+ | <td>5</td> | ||
+ | <td>5</td> | ||
+ | <td>1</td> | ||
+ | <td>33</td> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Thermal protocol is following</p> | ||
+ | <h3>2STEP Cycle (Tm value ? 63C)</h3> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td><td>94</td><td>120</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td><td>98</td><td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td><td>68</td><td>30sec / 1kbp</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td><td>4</td><td>Hold</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | Cycle: sequence2~3 × (25~45) | ||
+ | |||
+ | <h3>3STEP Cycle (Tm value ? 63C)</h3> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td><td>94</td><td>120</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td><td>98</td><td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td><td>Tm</td><td>30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td><td>68</td><td>30sec / 1kbp</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5</td><td>4</td><td>Hold</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | Cycle: sequence2~4 × (25~45) | ||
+ | |||
+ | |||
+ | <h2>PCR Purification</h2> | ||
+ | <p>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd) | ||
+ | <br>Purification of PCR products</p> | ||
+ | |||
+ | |||
+ | <h2>Digestion</h2> | ||
+ | Mix the following reagents in PCR tube. | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Solution</th> | ||
+ | <td>DNA</td> | ||
+ | <td>RE1 10U/µL</td> | ||
+ | <td>RE2 10U/µL</td> | ||
+ | <td>Appropriate buffer</td> | ||
+ | <td>Total</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Volume (µL)</th> | ||
+ | <td>16</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>2</td> | ||
+ | <td>20</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sequence</th><th>Temp. (°C)</th><th>Time (min)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td><td>37</td><td>120</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td><td>65</td><td>15</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td><td>4</td><td>Hold</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | <h2>Ligation</h2> | ||
+ | <p> | ||
+ | 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. | ||
+ | </p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Solution</th><td>Vector DNA</td><td>Insert DNA</td><td>DW</td><td>Mighty Mix</td><td>Total</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Volume (µL)</th><td>1</td><td>2</td><td>2</td><td>5</td><td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Thermal protocol is following</p> | ||
+ | <table | ||
+ | <tr> | ||
+ | <th>Sequence</th><th>Temp. (°C)</th><th>Time (min)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td><td>16</td><td>30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td><td>65</td><td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td><td>4</td><td>Hold</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | |||
+ | <h2>Electrophoresis</h2> | ||
+ | <ol> | ||
+ | <li>Put gel into electrophoresis tank.</li> | ||
+ | <li>Pore 2x TBE buffer into the tank to soak gel.</li> | ||
+ | <li>Add 5 µL of EtBr into cathod.</li> | ||
+ | <li>Pre-migration for 30 min at 100 V.</li> | ||
+ | <li>Apply DNA solution with 6x loading dye and ladder.</li> | ||
+ | <li>Start electrophoresis at 100 V.</li> | ||
+ | </ol> | ||
+ | |||
+ | |||
+ | <h2>Gel Extraction</h2> | ||
+ | <p>FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co., Ltd) | ||
+ | <br>DNA extraction from gel</p> | ||
+ | |||
+ | |||
+ | <h2>Ethanol precipitation</h2> | ||
+ | <ol> | ||
+ | <li>Add 1/10 volume of NaOAc, and 5/2 of 100% ethanol.</li> | ||
+ | <li>Leave it at room temperature for 5 min.</li> | ||
+ | <li>Centrifuge at 15,000 rpm for 15 min at 25°C.</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>Remove supernatant and air-dry at room temperature with light sheilding.</li> | ||
+ | <li>Suspend with 10 µL of TE.</li> | ||
+ | </ol> | ||
+ | |||
+ | |||
+ | <h2>Colony PCR</h2> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Solution</th> | ||
+ | <td>DNA</td> | ||
+ | <td>Kapa-Taq (Taq polymerase)</td> | ||
+ | <td>EX-F primer 10µM</td> | ||
+ | <td>PS-R primer 10µM</td> | ||
+ | <td>Total</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Volume (µL)</th> | ||
+ | <td>4.2</td> | ||
+ | <td>5</td> | ||
+ | <td>0.4</td> | ||
+ | <td>0.4</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td><td>94</td><td>120</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td><td>94</td><td>30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td><td>68</td><td>60 sec / 1kbp</td> | ||
+ | </tr> | ||
+ | <td>4</td><td>4</td><td>Hold</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | cycles: sequence2~3 × 25~45 | ||
+ | |||
+ | |||
+ | <h2>Sequencing</h2> | ||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Solution</th> | ||
+ | <td>5 x Sequencing Buffer</td> | ||
+ | <td>primer 1µL</td> | ||
+ | <td>template DNA</td> | ||
+ | <td>Ready Reaction Premix</td> | ||
+ | <td>DW</td> | ||
+ | <td>Total</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Volume (µL)</th> | ||
+ | <td>1.5</td> | ||
+ | <td>1.5</td> | ||
+ | <td>1</td> | ||
+ | <td>1</td> | ||
+ | <td>5</td> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | |||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td><td>96</td><td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td><td>50</td><td>5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td><td>60</td><td>240</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td><td>4</td><td>Hold</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | Cycle: sequence2~4 × 25 | ||
+ | |||
+ | |||
+ | <h3>Ethanol precipitation</h3> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Solution</th> | ||
+ | <td>PCR product</td> | ||
+ | <td>DW</td> | ||
+ | <td>3M NaOAc</td> | ||
+ | <td>Glycogen</td> | ||
+ | <td>100% EtOH</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Volume (µL)</th> | ||
+ | <td>10</td> | ||
+ | <td>10</td> | ||
+ | <td>2</td> | ||
+ | <td>1</td> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <ol> | ||
+ | <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>centrifuge at 15,000 rpm for 10 min at room temprature</li> | ||
+ | <li>Remove supernatant and air dry at room temperature, after that 10 µL of DW is added and dissolve the precipitate.</li> | ||
+ | <li>Electrophoresis</li> | ||
+ | <li>Resuspend the pellet to HiDi formamide and remove to 96-well plate.</li> | ||
+ | <li>Set the plate and start electrophoresis.</li> | ||
+ | </ol> | ||
+ | |||
+ | |||
+ | |||
+ | <h2>Competent Cells</h2> | ||
+ | <ol> | ||
+ | <li>Thaw original competent cells on ice.</li> | ||
+ | <li>Add 5 µL of original competent cells to 2 mL of LB.</li> | ||
+ | <li>Incubate the cells for 16 hrs at 37°C.</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>Take 50 mL of incubated cells to two differnt 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>Bring them to a one tube and centrifuge at 3,000 rpm for 20 min at 4°C.</li> | ||
+ | <li>Remove supernatant and add 32 mL of TB.</li> | ||
+ | <li>Add 32 µL of DMSO 10 times.</li> | ||
+ | <li>Take 50 µL and freeze with liquid nitrogen.</li> | ||
+ | </ol> | ||
+ | |||
+ | <h2>Transformation</h2> | ||
+ | <ol> | ||
+ | <li>Add plasmid to thawed competent cells on ice.</li> | ||
+ | <li>Incubate on ice for 30 min.</li> | ||
+ | <li>Add LB.</li> | ||
+ | <li>(Incubate the cells for 2 hrs at 37C.)</li> | ||
+ | <li>Spread 300 µL of the culture onto plate with LB and appropriate antibiotics.</li> | ||
+ | <li>Incubate the plate(s) at 37C for 16~20 hours.</li> | ||
+ | </ol> | ||
+ | |||
+ | |||
+ | |||
+ | <h2>Liquid Culture</h2> | ||
+ | <table> | ||
+ | <tr><th>Reagent</th><th>Volume</th></tr> | ||
+ | <tr><td>Single Colony</td><td>-</td></tr> | ||
+ | <tr><td>media</td><td>volume</td></tr> | ||
+ | <tr><td>antibiotic</td><td>volume</td></tr> | ||
+ | </table> | ||
+ | <p>Culture for several hrs.</p> | ||
+ | |||
+ | |||
+ | |||
+ | <h2>Mini-prep</h2> | ||
+ | <p>FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co., Ltd) | ||
+ | <br>fast / standard / low copy protocol</p> | ||
+ | |||
+ | |||
+ | <h2>Streaking (Single colony isolation)</h2> | ||
+ | <ol> | ||
+ | <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>Re-sterilise the loop and drag it across again.</li> | ||
+ | <li>Repeat method 3.</li> | ||
+ | </ol> | ||
+ | |||
+ | <h2>PEG precipitation</h2> | ||
+ | <ol> | ||
+ | <li>Add 13 µL of PEG to 20 µL of product(s).</li> | ||
+ | <li>Leave it at room temperature for 1 hr.</li> | ||
+ | <li>Centrifuge at 15,000 rpm for 20 min at 4°C.</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>Remove supernatant and air-dry at room temperature with light sheilding.</li> | ||
+ | <li>Suspend with 10 µL of TE.</li> | ||
+ | </ol> | ||
+ | |||
+ | |||
+ | <h2>Gel Free System</h2> | ||
+ | |||
+ | <h3>Preparation of biotinylated DNA fragments</h3> | ||
+ | <p> | ||
+ | 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. | ||
+ | </p> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Solution</th> | ||
+ | <td>template DNA</td> | ||
+ | <td>5'-biotinylated primer-F 10µM</td> | ||
+ | <td>5'-biotinylated Primer-R 10µM</td> | ||
+ | <td>MgSO<sub>4</sub></td> | ||
+ | <td>dNTPs</td> | ||
+ | <td>10x Buffer</td> | ||
+ | <td>KOD Plus Neo</td> | ||
+ | <td>DW</td> | ||
+ | <td>Total</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th>Volume (µL)</th> | ||
+ | <td>1</td> | ||
+ | <td>1.5</td> | ||
+ | <td>1.5</td> | ||
+ | <td>3</td> | ||
+ | <td>5</td> | ||
+ | <td>5</td> | ||
+ | <td>1</td> | ||
+ | <td>32</td> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>Thermal protocol is following</p> | ||
+ | <h3>2STEP Cycle (Tm value ? 63C)</h3> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>1</td><td>94</td><td>120</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>2</td><td>98</td><td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>3</td><td>68</td><td>30sec / 1kbp</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>4</td><td>4</td><td>Hold</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | Cycle: sequence2~3 × (25~45) | ||
+ | |||
+ | <h3>Preparation of magnetic beads</h3> | ||
+ | <ol> | ||
+ | <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> | ||
+ | <li>Remove supernatant.<li> | ||
+ | </ol> | ||
+ | |||
+ | <h3>Fixation to magnetic beads</h3> | ||
+ | <ol> | ||
+ | <li>Add 3 µL of PCR product (0.48 pmol) and 7 µL of TE to beads.</li> | ||
+ | <li>Mix by vibration using sonic-toothbrush.</li> | ||
+ | <li>Collect the beads using magnet.</li> | ||
+ | <li>Remove supernatant containing excess amount of free DNA fragment.</li> | ||
+ | </ol> | ||
+ | |||
+ | <h3>Double restriction digestion<h3> | ||
+ | <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>Mix by pumping using pipette.</li> | ||
+ | <li>Incubate at 37 °C for 30 min.</li> | ||
+ | <li>Collect the beads using magnet.</li> | ||
+ | <li>Obtain supernatant containing digested DNA fragment.</li> | ||
+ | <li>Purify the supernatant by ethanol precipitation.</li> | ||
+ | </ol> | ||
</html> | </html> |
Revision as of 02:40, 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 ? 63C)
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 98 | 10 |
3 | 68 | 30sec / 1kbp |
4 | 4 | Hold |
3STEP Cycle (Tm value ? 63C)
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 1µL | 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, after that 10 µL of DW is added and dissolve the precipitate.
- Electrophoresis
- 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 LB.
- (Incubate the cells for 2 hrs at 37C.)
- Spread 300 µL of the culture onto plate with LB and appropriate antibiotics.
- Incubate the plate(s) at 37C for 16~20 hours.
Liquid Culture
Reagent | Volume |
---|---|
Single Colony | - |
media | volume |
antibiotic | volume |
Culture for several 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 primer-F 10µM | 5'-biotinylated Primer-R 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 ? 63C)
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 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 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.