Dairyu6115 (Talk | contribs) |
|||
Line 35: | Line 35: | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Solution</th> | <th>Solution</th> | ||
<td>template DNA</td> | <td>template DNA</td> | ||
Line 47: | Line 47: | ||
<td>Total</td> | <td>Total</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<th>Volume (µL)</th> | <th>Volume (µL)</th> | ||
<td>1</td> | <td>1</td> | ||
Line 63: | Line 63: | ||
<h2>2STEP Cycle (Tm value > 63°C)</h2> | <h2>2STEP Cycle (Tm value > 63°C)</h2> | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>94</td><td>120</td> | <td>1</td><td>94</td><td>120</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>98</td><td>10</td> | <td>2</td><td>98</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>3</td><td>68</td><td>30sec / 1kbp</td> | <td>3</td><td>68</td><td>30sec / 1kbp</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>4</td><td>4</td><td>Hold</td> | <td>4</td><td>4</td><td>Hold</td> | ||
</tr> | </tr> | ||
Line 83: | Line 83: | ||
<h2>3STEP Cycle (Tm value < 63°C)</h2> | <h2>3STEP Cycle (Tm value < 63°C)</h2> | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>94</td><td>120</td> | <td>1</td><td>94</td><td>120</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>98</td><td>10</td> | <td>2</td><td>98</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>3</td><td>Tm</td><td>30</td> | <td>3</td><td>Tm</td><td>30</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>4</td><td>68</td><td>30sec / 1kbp</td> | <td>4</td><td>68</td><td>30sec / 1kbp</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>5</td><td>4</td><td>Hold</td> | <td>5</td><td>4</td><td>Hold</td> | ||
</tr> | </tr> | ||
Line 106: | Line 106: | ||
<h2>STEP DOWN</h2> | <h2>STEP DOWN</h2> | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>94</td><td>120</td> | <td>1</td><td>94</td><td>120</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>98</td><td>10</td> | <td>2</td><td>98</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>3</td><td>74</td><td>30sec / 1kbp</td> | <td>3</td><td>74</td><td>30sec / 1kbp</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>4</td><td>98</td><td>10</td> | <td>4</td><td>98</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>5</td><td>72</td><td>30</td> | <td>5</td><td>72</td><td>30</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>6</td><td>98</td><td>10</td> | <td>6</td><td>98</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>7</td><td>70</td><td>30</td> | <td>7</td><td>70</td><td>30</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>8</td><td>98</td><td>10</td> | <td>8</td><td>98</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>9</td><td>68</td><td>30</td> | <td>9</td><td>68</td><td>30</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>10</td><td>68</td><td>420</td> | <td>10</td><td>68</td><td>420</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>11</td><td>4</td><td>Hold</td> | <td>11</td><td>4</td><td>Hold</td> | ||
</tr> | </tr> | ||
Line 205: | Line 205: | ||
Mix the following reagents in PCR tube. | Mix the following reagents in PCR tube. | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Solution</th> | <th>Solution</th> | ||
<td>DNA</td> | <td>DNA</td> | ||
Line 213: | Line 213: | ||
<td>Total</td> | <td>Total</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<th>Volume (µL)</th> | <th>Volume (µL)</th> | ||
<td>16</td> | <td>16</td> | ||
Line 223: | Line 223: | ||
</table> | </table> | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (min)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (min)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>37</td><td>120</td> | <td>1</td><td>37</td><td>120</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>65</td><td>15</td> | <td>2</td><td>65</td><td>15</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>3</td><td>4</td><td>Hold</td> | <td>3</td><td>4</td><td>Hold</td> | ||
</tr> | </tr> | ||
Line 264: | Line 264: | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Solution</th><td>Vector DNA</td><td>Insert DNA</td><td>DW</td><td>Mighty Mix</td><td>Total</td> | <th>Solution</th><td>Vector DNA</td><td>Insert DNA</td><td>DW</td><td>Mighty Mix</td><td>Total</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<th>Volume (µL)</th><td>1</td><td>2</td><td>2</td><td>5</td><td>10</td> | <th>Volume (µL)</th><td>1</td><td>2</td><td>2</td><td>5</td><td>10</td> | ||
</tr> | </tr> | ||
Line 273: | Line 273: | ||
Thermal protocol is following | Thermal protocol is following | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (min)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (min)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>16</td><td>30</td> | <td>1</td><td>16</td><td>30</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>65</td><td>10</td> | <td>2</td><td>65</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>3</td><td>4</td><td>Hold</td> | <td>3</td><td>4</td><td>Hold</td> | ||
</tr> | </tr> | ||
Line 403: | Line 403: | ||
<span class="nomal2"> | <span class="nomal2"> | ||
<table> | <table> | ||
− | + | ||
<tr align="center"> | <tr align="center"> | ||
− | + | ||
<th>Solution</th> | <th>Solution</th> | ||
<td>DNA</td> | <td>DNA</td> | ||
Line 424: | Line 424: | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>94</td><td>120</td> | <td>1</td><td>94</td><td>120</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>94</td><td>30</td> | <td>2</td><td>94</td><td>30</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>3</td><td>68</td><td>60 sec / 1kbp</td> | <td>3</td><td>68</td><td>60 sec / 1kbp</td> | ||
</tr> | </tr> | ||
Line 465: | Line 465: | ||
<span class="nomal2"> | <span class="nomal2"> | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Solution</th> | <th>Solution</th> | ||
<td>5 x Sequencing Buffer</td> | <td>5 x Sequencing Buffer</td> | ||
Line 474: | Line 474: | ||
<td>Total</td> | <td>Total</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<th>Volume (µL)</th> | <th>Volume (µL)</th> | ||
<td>1.5</td> | <td>1.5</td> | ||
Line 486: | Line 486: | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>96</td><td>10</td> | <td>1</td><td>96</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>50</td><td>5</td> | <td>2</td><td>50</td><td>5</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>3</td><td>60</td><td>240</td> | <td>3</td><td>60</td><td>240</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>4</td><td>4</td><td>Hold</td> | <td>4</td><td>4</td><td>Hold</td> | ||
</tr> | </tr> | ||
Line 507: | Line 507: | ||
<h2>Ethanol precipitation</h2> | <h2>Ethanol precipitation</h2> | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Solution</th> | <th>Solution</th> | ||
<td>PCR product</td> | <td>PCR product</td> | ||
Line 515: | Line 515: | ||
<td>100% EtOH</td> | <td>100% EtOH</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<th>Volume (µL)</th> | <th>Volume (µL)</th> | ||
<td>10</td> | <td>10</td> | ||
Line 713: | Line 713: | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Solution</th> | <th>Solution</th> | ||
<td>template DNA</td> | <td>template DNA</td> | ||
Line 725: | Line 725: | ||
<td>Total</td> | <td>Total</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<th>Volume (µL)</th> | <th>Volume (µL)</th> | ||
<td>1</td> | <td>1</td> | ||
Line 741: | Line 741: | ||
<h2>2STEP Cycle (Tm value > 63°C)</h2> | <h2>2STEP Cycle (Tm value > 63°C)</h2> | ||
<table> | <table> | ||
− | <tr> | + | <tr align="center"> |
<th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | <th>Sequence</th><th>Temp. (°C)</th><th>Time (sec)</th> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>1</td><td>94</td><td>120</td> | <td>1</td><td>94</td><td>120</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>2</td><td>98</td><td>10</td> | <td>2</td><td>98</td><td>10</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>3</td><td>68</td><td>30sec / 1kbp</td> | <td>3</td><td>68</td><td>30sec / 1kbp</td> | ||
</tr> | </tr> | ||
− | <tr> | + | <tr align="center"> |
<td>4</td><td>4</td><td>Hold</td> | <td>4</td><td>4</td><td>Hold</td> | ||
</tr> | </tr> |
Revision as of 13:36, 17 October 2016
Team:HokkaidoU Japan
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
Cycles: sequence2~3 × 25~45
Cycle: sequence2~4 × 25
Thermal protocol is following
Cycle: sequence2~3 × (25~45)
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: sequence2~3 × (25~45)
Cycle: sequence2~4 × (25~45)
Cycle:
sequence2~3 × 5
sequence4~5 × 5
sequence6~7 × 5
sequence8~9 × 15
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 |
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 |
STEP DOWN
Sequence | Temp. (°C) | Time (sec) |
---|---|---|
1 | 94 | 120 |
2 | 98 | 10 |
3 | 74 | 30sec / 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 |
sequence2~3 × 5
sequence4~5 × 5
sequence6~7 × 5
sequence8~9 × 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 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.
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.
- 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
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 (Taqpolymerase) | 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
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 |
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.