Team:HokkaidoU Japan/Notebook/Protocols

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<h1>Protocols</h1>
<h1>Protocols</h1>
<h2>Transformation</h2>
<h2>Transformation</h2>
<ol>
<ol>
-
   <li>Added (1~2)  &micro;L  of (DNA) to (50)  &micro;L  of thawed competent cells on ice.</li>
+
   <li>Add (1~5)  &micro;L  of (DNA) to (50)  &micro;L  of thawed competent cells on ice.</li>
-
   <li>Incubated on ice for 30 min.</li>
+
   <li>Incubate on ice for 30 min.</li>
-
   <li>Added (600)  &micro;L  of LB.</li>
+
   <li>Add (600)  &micro;L  of LB.</li>
-
   <li>(Incubated the cells for 2 hrs at 37C.)</li>
+
   <li>(Incubate the cells for 2 hrs at 37C.)</li>
   <li>Spread 300  &micro;L  of the culture onto plate with LB and appropriate antibiotics.</li>
   <li>Spread 300  &micro;L  of the culture onto plate with LB and appropriate antibiotics.</li>
-
   <li>(Added 900  &micro;L  of LB to 100  &micro;L  of the culture and spread 300  &micro;L  of it onto second plate.)</li>
+
   <li>(Add 900  &micro;L  of LB to 100  &micro;L  of the culture and spread 300  &micro;L  of it onto second plate.)</li>
-
   <li>Incubated the plate(s) at 37C for 16~20 hours.</li>
+
   <li>Incubate the plate(s) at 37C for 16~20 hours.</li>
</ol>
</ol>
<h2>Mini-prep</h2>
<h2>Mini-prep</h2>
<p>
<p>
-
Used mini-prep kit of Nippon genetics: FastGene Plasmid mini kit. This kit contains these reagents and wares: mP1 (resuspension buffer), mP2 (lysis buffer), mP3 (neutralization buffer), mP4 (first membrane wash buffer), mP5(second membrane wash buffer), mP6(elution buffer), column and collection tube.
+
  Use FastGene<sup>TM</sup> Plasmid mini kit (Nippon Genetics Co.,Ltd). This kit contains these reagents and wares: mP1 (resuspension buffer), mP2 (lysis buffer), mP3 (neutralization buffer), mP4 (first membrane wash buffer), mP5(second membrane wash buffer), mP6(elution buffer), column and collection tube.
</p>
</p>
<ol>
<ol>
-
<li>Centrifuged (1~5) mL of culture at (over 10,000) rpm for 2 min.</li>
+
  <li>Centrifuge (1~5) mL of culture at (over 10,000) rpm for 2 min.</li>
-
<li>Removed the supernatant.</li>
+
  <li>Remove the supernatant.</li>
-
<li>Added 200  &micro;L  of mP1 and voltexed it.</li>
+
  <li>Add 200  &micro;L  of mP1 and voltex it.</li>
-
<li>Added 200  &micro;L  of mP2 and inverted the tube then left it for 2 min at room temperature.</li>
+
  <li>Add 200  &micro;L  of mP2 and invert the tube then leave it for 2 min at room temperature.</li>
-
<li>Added 300  &micro;L  of mP3 then inverted the tube.</li>
+
  <li>Add 300  &micro;L  of mP3 then invert the tube.</li>
-
<li>Centrifuged at 13,000 rpm for 2 min.</li>
+
  <li>Centrifuge at 13,000 rpm for 2 min.</li>
-
<li>Loaded the supernatant to column tube.</li>
+
  <li>Load the supernatant to column tube.</li>
-
<li>Centrifuged at 13,000 rpm for 1 min.</li>
+
  <li>Centrifuge at 13,000 rpm for 1 min.</li>
-
<li>Removed filtrate and added 400  &micro;L  of mP4 then centrifuged 13,000 rpm for 1 min.</li>
+
  <li>Remove filtrate and add 400  &micro;L  of mP4 then centrifuge 13,000 rpm for 1 min.</li>
-
<li>Removed filtrate and added 600  &micro;L  of mP5 then centrifuged 13,000 rpm for 1 min.</li>
+
  <li>Remove filtrate and add 600  &micro;L  of mP5 then centrifuge 13,000 rpm for 1 min.</li>
-
<li>Removed filtrate and centrifuged 13,000 rpm for 2 min.</li>
+
  <li>Remove filtrate and centrifuge 13,000 rpm for 2 min.</li>
-
<li>Set column into 1.5 mL tube and added 50  &micro;L  of mP6.</li>
+
  <li>Set column into 1.5 mL tube and add 50  &micro;L  of mP6.</li>
-
<li>Centrifuged at 13,000 rpm for 2 min.</li>
+
  <li>Centrifuge at 13,000 rpm for 2 min.</li>
</ol>
</ol>
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<h2>Ethanol precipitation</h2>
<h2>Ethanol precipitation</h2>
<ol>
<ol>
-
   <li>Added (5)  &micro;L  of NaOAc, 1.5  &micro;L  of glycogen and (125)  &micro;L  of 100% ethanol.</li>
+
   <li>Add (5)  &micro;L  of NaOAc, 1.5  &micro;L  of glycogen and (125)  &micro;L  of 100% ethanol.</li>
-
   <li>(Left it at -80C for 1 hr. / Soaked liquid nitrogen in an instant.)</li>
+
   <li>(Leave it at -80C for 1 hr. / Soak liquid nitrogen in an instant.)</li>
-
   <li>Centrifuged at 15,000 rpm for (10~15) min at 4C.</li>
+
   <li>Centrifuge at 15,000 rpm for (10~15) min at 4C.</li>
-
   <li>Removed supernatant and added (220)  &micro;L  of 70% ethanol.</li>
+
   <li>Remove supernatant and add (220)  &micro;L  of 70% ethanol.</li>
-
   <li>Centrifuged at 15,000 rpm for (5~15) min at 4C.</li>
+
   <li>Centrifuge at 15,000 rpm for (5~15) min at 4C.</li>
-
   <li>Removed supernatant and air-dried at room temperature with light sheilding.</li>
+
   <li>Remove supernatant and air-dry at room temperature with light sheilding.</li>
-
   <li>Suspended with 10  &micro;L  of DW.</li>
+
   <li>Suspend with 10  &micro;L  of DW.</li>
</ol>
</ol>
<h2>Ligation</h2>
<h2>Ligation</h2>
<p>
<p>
-
Mixed the following reagents in 0.2 mL PCR tube. Used Ligation Mighty Mix (TAKARA BIO INC.) which contains ligase and buffer.
+
  Mix the following reagents in 0.2 mL PCR tube. Use DNA Ligation Kit &lt;Mighty Mix&rt; (Takara Bio Inc.) which contains ligase and buffer.
</p>
</p>
<table>
<table>
   <tr>
   <tr>
-
     <th>Solution</th><td>Vector DNA</td><td>Insert DNA</td><td>DW</td><td>Ligation 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>
Line 86: Line 90:
<h2>Digestion</h2>
<h2>Digestion</h2>
-
Mixed the following reagents in PCR tube.
+
Mix the following reagents in PCR tube.
<table>
<table>
   <tr>
   <tr>
     <th>Solution</th>
     <th>Solution</th>
     <td>DNA</td>
     <td>DNA</td>
-
     <td>Restriction enzyme 1 10U/&micro;L</td>
+
     <td>RE1 10U/&micro;L</td>
-
     <td>Restriction enzyme 2 10U/&micro;L</td>
+
     <td>RE2 10U/&micro;L</td>
     <td>Appropriate buffer</td>
     <td>Appropriate buffer</td>
-
    <td>DW</td>
 
     <td>Total</td>
     <td>Total</td>
   </tr>
   </tr>
   <tr>
   <tr>
     <th>Volume (&micro;L)</th>
     <th>Volume (&micro;L)</th>
-
     <td></td>
+
     <td>16</td>
-
     <td></td>
+
     <td>1</td>
-
     <td></td>
+
     <td>1</td>
-
     <td></td>
+
     <td>2</td>
-
     <td></td>
+
     <td>20</td>
-
    <td></td>
+
   </tr>
   </tr>
</table>
</table>
Line 125: Line 127:
<ol>
<ol>
   <li>Put gel into electrophoresis tank.</li>
   <li>Put gel into electrophoresis tank.</li>
-
   <li>Pored 2x TBE buffer into the tank to soak gel.</li>
+
   <li>Pore 2x TBE buffer into the tank to soak gel.</li>
-
   <li>Added 5  &micro;L  of EtBr into cathod.</li>
+
   <li>Add 5  &micro;L  of EtBr into cathod.</li>
   <li>Pre-migration for 30 min at 100 V.</li>
   <li>Pre-migration for 30 min at 100 V.</li>
-
   <li>Applied DNA solution with 6x loading dye and ladder.</li>
+
   <li>Apply DNA solution with 6x loading dye and ladder.</li>
-
   <li>Started electrophoresis at 100 V.</li>
+
   <li>Start electrophoresis at 100 V.</li>
</ol>
</ol>
Line 135: Line 137:
<h2>Gel extraction</h2>
<h2>Gel extraction</h2>
<p>
<p>
-
   Used Gel extraction kit of Nippon genetics: FastGene Gel/PCR Extraction kit. This kit contains these reagents and wares: GP1 (binding buffer), GP2 (wash buffer), GP3 (elution buffer), column and collection tube.
+
   Use FastGene<sup>TM</sup> Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd). This kit contains these reagents and wares: GP1 (binding buffer), GP2 (wash buffer), GP3 (elution buffer), column and collection tube.
</p>
</p>
<ol>
<ol>
-
   <li>Added 500  &micro;L  of GP1 to (~300 mg of) migrated gel and vortex.</li>
+
   <li>Add 500  &micro;L  of GP1 to (~300 mg of) migrate gel and vortex.</li>
-
   <li>Incubated the mixture at 55C for (10~15) min and inverted it.</li>
+
   <li>Incubate the mixture at 55C for (10~15) min and inverted it.</li>
-
   <li>Loaded the sample onto the column.</li>
+
   <li>Load the sample onto the column.</li>
-
   <li>Centrifuged at 13,000 rpm for 1 min.</li>
+
   <li>Centrifuge at 13,000 rpm for 1 min.</li>
-
   <li>Removed filtrate and added 600  &micro;L  of GP2 and centrifuged at 13,000 rpm for 1 min.</li>
+
   <li>Remove filtrate and add 600  &micro;L  of GP2 and centrifuge at 13,000 rpm for 1 min.</li>
-
   <li>Repeated step 5.</li>
+
   <li>Repeat step 5.</li>
-
   <li>Removed filtrate and centrifuged at 13,000 rpm for 2 min.</li>
+
   <li>Remove filtrate and centrifuge at 13,000 rpm for 2 min.</li>
-
   <li>Set column into 1.5 mL tube and added 50  &micro;L  of GP6.</li>
+
   <li>Set column into 1.5 mL tube and add 50  &micro;L  of GP6.</li>
-
   <li>Centrifuged at 13,000 rpm for 2 min.</li>
+
   <li>Centrifuge at 13,000 rpm for 2 min.</li>
</ol>
</ol>
Line 152: Line 154:
<h2>PCR</h2>
<h2>PCR</h2>
<p>
<p>
-
   Used KOD-Plus-Neo (TOYOBO) as polymerase. Mixed PCR solutions and ran the PCR machine in a program which is detailed below.
+
   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>
</p>
<table>
<table>
Line 158: Line 160:
     <th>Solution</th>
     <th>Solution</th>
     <td>template DNA</td>
     <td>template DNA</td>
-
     <td>Forward Primer 10&micro;M</td>
+
     <td>Primer-F 10&micro;M</td>
-
     <td>Reverse Primer 10&micro;M</td>
+
     <td>Primer-R 10&micro;M</td>
     <td>MgSO<sub>4</sub></td>
     <td>MgSO<sub>4</sub></td>
     <td>dNTPs</td>
     <td>dNTPs</td>
-
     <td>10x KOD-Plus-Neo Buffer</td>
+
     <td>10x Buffer</td>
-
     <td>KOD-Plus-Neo</td>
+
     <td>KOD Plus Neo</td>
     <td>DW</td>
     <td>DW</td>
     <td>Total</td>
     <td>Total</td>
Line 181: Line 183:
</table>
</table>
<p>Thermal protocol is following</p>
<p>Thermal protocol is following</p>
 +
<h3>2STEP Cycle (Tm value ≥ 63C)</h3>
<table>
<table>
   <tr>
   <tr>
-
     <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (min)</th>
+
     <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>1</td><td>16</td><td>30</td>
+
     <td>1</td><td>94</td><td>120</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>2</td><td>65</td><td>10</td>
+
     <td>2</td><td>98</td><td>10</td>
   </tr>
   </tr>
   <tr>
   <tr>
-
     <td>3</td><td>4</td><td>Hold</td>
+
     <td>3</td><td>68</td><td>30sec / 1kbp</td>
 +
  </tr>
 +
  <tr>
 +
    <td>4</td><td>4</td><td>Hold</td>
   </tr>
   </tr>
</table>
</table>
 +
Cycle: sequence2~3 &times; (25~45)
-
2STEP Cycle (Tm value 63C)
+
<h3>3STEP Cycle (Tm value 63C)</h3>
-
Number
+
<table>
-
Temp.
+
  <tr>
-
Second
+
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
-
1
+
  </tr>
-
94
+
  <tr>
-
120
+
    <td>1</td><td>94</td><td>120</td>
-
2
+
  </tr>
-
98
+
  <tr>
-
10
+
    <td>2</td><td>98</td><td>10</td>
-
3
+
  </tr>
-
68
+
  <tr>
-
(30/kbp)
+
    <td>3</td><td>Tm</td><td>30</td>
-
4
+
  </tr>
-
4
+
  <tr>
-
HOLD
+
    <td>4</td><td>68</td><td>30sec / 1kbp</td>
-
Cycle: 2~3 x (25~45)
+
  </tr>
 +
  <tr>
 +
    <td>5</td><td>4</td><td>Hold</td>
 +
  </tr>
 +
</table>
 +
Cycle: sequence2~4 &times; (25~45)
-
 
+
<h2>Sequencing</h2>
-
3STEP Cycle (Tm value ≤ 63C)
+
-
Number
+
-
Temp.
+
-
Second
+
-
1
+
-
94
+
-
120
+
-
2
+
-
98
+
-
10
+
-
3
+
-
(Tm value of primer)
+
-
30
+
-
4
+
-
68
+
-
(30/kbp)
+
-
5
+
-
4
+
-
HOLD
+
-
Cycle: 2~4 x (25~45)
+
-
 
+
-
 
+
-
cycleの右側の数字の意味がわかりにくいなと思いました
+
-
Sequencing
+
<table>
<table>
   <tr>
   <tr>
     <th>Solution</th>
     <th>Solution</th>
 +
    <td>5 x Sequencing Buffer</td>
 +
    <td>primer 1&micro;L</td>
     <td>template DNA</td>
     <td>template DNA</td>
-
     <td>Forward Primer</td>
+
     <td>Ready Reaction Premix</td>
-
    <td>Reverse </td>
+
     <td>DW</td>
-
     <td></td>
+
     <td>Total</td>
     <td>Total</td>
   </tr>
   </tr>
   <tr>
   <tr>
     <th>Volume (&micro;L)</th>
     <th>Volume (&micro;L)</th>
-
     <td></td>
+
     <td>1.5</td>
-
     <td></td>
+
     <td>1.5</td>
-
     <td></td>
+
     <td>1</td>
-
     <td></td>
+
     <td>1</td>
-
     <th></th>
+
     <td>5</td>
 +
    <td>10</td>
   </tr>
   </tr>
</table>
</table>
-
Solution
 
-
Volume( &micro;L )
 
-
5 x Sequencing Buffer 
 
-
1.5
 
-
( primer) 1 uM
 
-
1.5
 
-
template DNA
 
-
1
 
-
Ready Reaction Premix
 
-
1
 
-
DW
 
-
5
 
-
Total
 
-
10
 
 +
<table>
 +
  <tr>
 +
    <th>Sequence</th><th>Temp. (&deg;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 &times; 25
-
Number
 
-
Temp.
 
-
Second
 
-
1
 
-
96
 
-
10
 
-
2
 
-
50
 
-
5
 
-
3
 
-
60
 
-
240
 
-
5
 
-
4
 
-
HOLD
 
-
Cycle: 2 x 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 (&micro;L)</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 &micro;L  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  &micro;L  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>
-
Ethanol Presipitation
+
<h2>Streaking (Single colony isolation)</h2>
-
 
+
<ol>
-
Solution
+
  <li>Pick the colony with an inoculating loop from the agar plate.</li>
-
Volume( &micro;L )
+
  <li>Drag the loop across on a new agar plate.</li>
-
PCR product  
+
  <li>Re-sterilise the loop and drag it across again.</li>
-
10
+
  <li>Repeat method 3.</li>
-
DW
+
</ol>
-
10
+
-
3 M NaOAc  
+
-
2
+
-
Glycogen   
+
-
1
+
-
100% EtOH
+
-
50
+
-
centrifuged at 15,000 rpm for 15 min at room temprature
+
-
Removed supernatant ,added 100 &micro;L  of 70% EtOH and tap tubes by finger.
+
-
centrifuged at 15,000 rpm for 10 min at room temprature
+
-
Removed supernatant and air dried at room temperature, after that 10  &micro;L  of  DW was added and dissolved the precipitate.
+
-
Electrophoresis
+
-
Resuspended the pellet to HiDi formamide and removed to 96-well plate.
+
-
Set the plate and started electrophoresis.
+
 +
<h2>Colony PCR</h2>
 +
<table>
 +
  <tr>
 +
    <th>Solution</th>
 +
    <td>DNA</td>
 +
    <td>Kapa-Taq (Taq polymerase)</td>
 +
    <td>EX-F primer 10&micro;M</td>
 +
    <td>PS-R primer 10&micro;M</td>
 +
    <td>DW</td>
 +
    <td>Total</td>
 +
  </tr>
 +
  <tr>
 +
    <th>Volume (&micro;L)</th>
 +
    <td>4</td>
 +
    <td>10</td>
 +
    <td>0.8</td>
 +
    <td>0.8</td>
 +
    <td>8.4</td>
 +
    <td>20</td>
 +
  </tr>
 +
</table>
-
Streaking (Single colony isolation)
+
<table>
-
1.  Pick the colony with an inoculating loop from the agar plate.
+
  <tr>
-
2.  Drag the loop across on a new agar plate.
+
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (sec)</th>
-
3.  Re-sterilise the loop and drag it across again.
+
  </tr>
-
4.  Repeat method 3.
+
  <tr>
-
 
+
    <td>1</td><td>95</td><td>120</td>
-
 
+
  </tr>
-
 
+
  <tr>
-
Colony PCR
+
    <td>2</td><td>95</td><td>30</td>
-
 
+
  </tr>
-
Solution
+
  <tr>
-
Volume( &micro;L )
+
    <td>3</td><td>68.9</td><td>30</td>
-
DNA solution(1 colony/10  &micro;L  DW)
+
  </tr>
-
4
+
  <tr>
-
Kapa-Taq(Taq polymerase)
+
    <td>4</td><td>72</td><td>60</td>
-
10
+
  </tr>
-
10 uM EX-F primer
+
  <tr>
-
0.8
+
    <td>5</td><td>72</td><td>120</td>
-
10 uM PS-R primer  
+
  </tr>
-
0.8
+
  <tr>
-
DW
+
    <td>6</td><td>4</td><td>Hold</td>
-
8.4
+
  </tr>
-
Total
+
</table>
-
20
+
cycles: sequence2~4 &times; 30~45
-
 
+
-
 
+
-
Number
+
-
Temp.
+
-
Second
+
-
1
+
-
95
+
-
120
+
-
2
+
-
95
+
-
30
+
-
3
+
-
68.9
+
-
30
+
-
4
+
-
72
+
-
60
+
-
5
+
-
72
+
-
120
+
-
6
+
-
4
+
-
HOLD
+
-
2~4: 30~45 cycles
+
Line 374: Line 363:
   <li>Add 50  &micro;L  of Standard Dilution Buffer to 13-well(changeable) of a 96-well plate, and make a standard curve.</li>
   <li>Add 50  &micro;L  of Standard Dilution Buffer to 13-well(changeable) of a 96-well plate, and make a standard curve.</li>
</ol>
</ol>
-
&beta;-Gal Standard
+
<table>
-
(milliunits)
+
  <tr>
-
Standard Dilution Buffer Volume
+
<th>&beta;-Gal Standard (milliunits)</th>
-
&beta;-Gal Standard Volume
+
<th>Standard Dilution Buffer Volume</th>
-
20
+
<th>&beta;-Gal Standard Volume</th>
-
999  &micro;L
+
</tr><tr>
-
1  &micro;L  of &beta;-gal standard stock
+
<td>20</td>
-
10
+
<td>999  &micro;L</td>
-
100  &micro;L
+
<td>1  &micro;L  of &beta;-gal standard stock</td>
-
100  &micro;L  of 20 mu &beta;-gal standard
+
</tr><tr>
-
5
+
<td>10</td>
-
100  &micro;L
+
<td>100  &micro;L</td>
-
100  &micro;L  of 10 mu &beta;-gal standard
+
<td>100  &micro;L  of 20 mu &beta;-gal standard</td>
-
2.5
+
</tr><tr>
-
100  &micro;L
+
<td>5</td>
-
100  &micro;L  of 5 mu &beta;-gal standard
+
<td>100  &micro;L</td>
-
1.25
+
<td>100  &micro;L  of 10 mu &beta;-gal standard</td>
-
100  &micro;L
+
</tr><tr>
-
100  &micro;L  of 2.5 mu &beta;-gal standard
+
<td>2.5</td>
-
0.625
+
<td>100  &micro;L</td>
-
100  &micro;L
+
<td>100  &micro;L  of 5 mu &beta;-gal standard</td>
-
100  &micro;L  of 1.25 mu &beta;-gal standard
+
</tr><tr>
-
0.3125
+
<td>1.25</td>
-
100  &micro;L
+
<td>100  &micro;L</td>
-
100  &micro;L  of 0.625 mu &beta;-gal standard
+
<td>100  &micro;L  of 2.5 mu &beta;-gal standard</td>
-
0.15625
+
</tr><tr>
-
100  &micro;L
+
<td>0.625</td>
-
100  &micro;L  of 0.3125 mu &beta;-gal standard
+
<td>100  &micro;L</td>
-
0.078125
+
<td>100  &micro;L  of 1.25 mu &beta;-gal standard</td>
-
100  &micro;L
+
</tr><tr>
-
100  &micro;L  of 0.15625 mu &beta;-gal standard
+
<td>0.3125</td>
-
0.0390625
+
<td>100  &micro;L</td>
-
100  &micro;L
+
<td>100  &micro;L  of 0.625 mu &beta;-gal standard</td>
-
100  &micro;L  of 0.078125 mu &beta;-gal standard
+
</tr><tr>
-
0.01953125
+
<td>0.15625</td>
-
100  &micro;L
+
<td>100  &micro;L</td>
-
100  &micro;L  of 0.0390625 mu &beta;-gal standard
+
<td>100  &micro;L  of 0.3125 mu &beta;-gal standard</td>
-
0.009765625
+
</tr><tr>
-
100  &micro;L
+
<td>0.078125</td>
-
100  &micro;L  of 0.01953125 mu &beta;-gal standard
+
<td>100  &micro;L</td>
 +
<td>100  &micro;L  of 0.15625 mu &beta;-gal standard</td>
 +
</tr><tr>
 +
<td>0.0390625</td>
 +
<td>100  &micro;L</td>
 +
<td>100  &micro;L  of 0.078125 mu &beta;-gal standard</td>
 +
</tr><tr>
 +
<td>0.01953125</td>
 +
<td>100  &micro;L</td>
 +
<td>100  &micro;L  of 0.0390625 mu &beta;-gal standard</td>
 +
</tr><tr>
 +
<td>0.009765625</td>
 +
<td>100  &micro;L</td>
 +
<td>100  &micro;L  of 0.01953125 mu &beta;-gal standard</td>
 +
</tr><tr>
 +
<td>0</td>
 +
<td>100  &micro;L</td>
 +
</table>
-
 
+
<ol>
-
1.  Add 50  &micro;L  of each sample to new well.
+
  <li>Add 50  &micro;L  of each sample to new well.</li>
-
2.  Add 50  &micro;L  of 1x CPRG Substrate Solution to each well and incubate the plate at room temperature for about 2 hrs.
+
  <li>Add 50  &micro;L  of 1x CPRG Substrate Solution to each well and incubate the plate at room temperature for about 2 hrs.</li>
-
3.  Add 100  &micro;L  of Stop Buffer to each well.
+
  <li>Add 100  &micro;L  of Stop Buffer to each well.</li>
-
4.  Monitor the color development(Measure the absorbance of each sample) at 570-595 nm.
+
  <li>Monitor the color development(Measure the absorbance of each sample) at 570-595 nm.</li>
-
5.  Calculate the expression levels(the activity) of lacZ based on a standard curve
+
  <li>Calculate the expression levels(the activity) of lacZ based on a standard curve</li>
 +
</ol>
 +
<h2>Golden Gate Assembly</h2>
 +
<p>
 +
  PCR insert DNA with BsaI-adding primer (please refer <a href="https://2013.igem.org/Team:HokkaidoU_Japan/Optimization/Primer_Designer">Primer Designer for Maestro</a>), purify the PCRed DNA and measure their concentrations.
 +
</p>
 +
<p>
 +
  Then, mix the following reagents in 0.2 mL PCR tube. Use BsaI / CutSmart<sup>TM</sup>, T4 DNA Ligase and its buffer (New England Biolabs Ltd). For vector DNA, use BBa_K1084501 etc..
 +
</p>
 +
<table>
 +
  <tr>
 +
    <th>Solution</th><td>BsaI</td><td>10&times; CutSmart buffer</td><td>T4 Ligase</td><td>10&times; T4 buffer</td><td>vector DNA</td><td>each insert DNA</td><td>DW</td><td>Total</td>
 +
  </tr>
 +
  <tr>
 +
    <th>Volume</th><td>1 &micro;L</td><td>1.5 &micro;L</td><td>1 &micro;L</td><td>1.5 &micro;L</td><td>100 ng</td><td>equimolar with vector DNA<td>for messing up</td><td>15 &micro;L</td>
 +
  </tr>
 +
</table>
 +
<p>Used PCR machine to digest and ligate in one-pot.</p>
 +
<table>
 +
  <tr>
 +
    <th>Sequence</th><th>Temp. (&deg;C)</th><th>Time (min)</th>
 +
  </tr>
 +
  <tr>
 +
    <td>1</td><td>37</td><td>3</td>
 +
  </tr>
 +
  <tr>
 +
    <td>2</td><td>16</td><td>4</td>
 +
  </tr>
 +
  <tr>
 +
    <td>3</td><td>50</td><td>5</td>
 +
  </tr>
 +
  <tr>
 +
    <td>4</td><td>80</td><td>5</td>
 +
  </tr>
 +
  <tr>
 +
    <td>5</td><td>4</td><td>Hold</td>
 +
  </tr>
 +
</table>
 +
cycles: sequence1~2 &times; 25
<!-- end contents / begin footer -->
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Latest revision as of 13:21, 28 October 2013

Maestro E.coli

Notebook

Protocols

Transformation

  1. Add (1~5) µL of (DNA) to (50) µL of thawed competent cells on ice.
  2. Incubate on ice for 30 min.
  3. Add (600) µL of LB.
  4. (Incubate the cells for 2 hrs at 37C.)
  5. Spread 300 µL of the culture onto plate with LB and appropriate antibiotics.
  6. (Add 900 µL of LB to 100 µL of the culture and spread 300 µL of it onto second plate.)
  7. Incubate the plate(s) at 37C for 16~20 hours.

Mini-prep

Use FastGeneTM Plasmid mini kit (Nippon Genetics Co.,Ltd). This kit contains these reagents and wares: mP1 (resuspension buffer), mP2 (lysis buffer), mP3 (neutralization buffer), mP4 (first membrane wash buffer), mP5(second membrane wash buffer), mP6(elution buffer), column and collection tube.

  1. Centrifuge (1~5) mL of culture at (over 10,000) rpm for 2 min.
  2. Remove the supernatant.
  3. Add 200 µL of mP1 and voltex it.
  4. Add 200 µL of mP2 and invert the tube then leave it for 2 min at room temperature.
  5. Add 300 µL of mP3 then invert the tube.
  6. Centrifuge at 13,000 rpm for 2 min.
  7. Load the supernatant to column tube.
  8. Centrifuge at 13,000 rpm for 1 min.
  9. Remove filtrate and add 400 µL of mP4 then centrifuge 13,000 rpm for 1 min.
  10. Remove filtrate and add 600 µL of mP5 then centrifuge 13,000 rpm for 1 min.
  11. Remove filtrate and centrifuge 13,000 rpm for 2 min.
  12. Set column into 1.5 mL tube and add 50 µL of mP6.
  13. Centrifuge at 13,000 rpm for 2 min.

Ethanol precipitation

  1. Add (5) µL of NaOAc, 1.5 µL of glycogen and (125) µL of 100% ethanol.
  2. (Leave it at -80C for 1 hr. / Soak liquid nitrogen in an instant.)
  3. Centrifuge at 15,000 rpm for (10~15) min at 4C.
  4. Remove supernatant and add (220) µL of 70% ethanol.
  5. Centrifuge at 15,000 rpm for (5~15) min at 4C.
  6. Remove supernatant and air-dry at room temperature with light sheilding.
  7. Suspend with 10 µL of DW.

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.

SolutionVector DNAInsert DNADWMighty MixTotal
Volume (µL)122510

Thermal protocol is following

SequenceTemp. (°C)Time (min)
11630
26510
34Hold

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
SequenceTemp. (°C)Time (min)
137120
26515
34Hold

Electrophoresis

  1. Put gel into electrophoresis tank.
  2. Pore 2x TBE buffer into the tank to soak gel.
  3. Add 5 µL of EtBr into cathod.
  4. Pre-migration for 30 min at 100 V.
  5. Apply DNA solution with 6x loading dye and ladder.
  6. Start electrophoresis at 100 V.

Gel extraction

Use FastGeneTM Gel/PCR Extraction kit (Nippon Genetics Co.,Ltd). This kit contains these reagents and wares: GP1 (binding buffer), GP2 (wash buffer), GP3 (elution buffer), column and collection tube.

  1. Add 500 µL of GP1 to (~300 mg of) migrate gel and vortex.
  2. Incubate the mixture at 55C for (10~15) min and inverted it.
  3. Load the sample onto the column.
  4. Centrifuge at 13,000 rpm for 1 min.
  5. Remove filtrate and add 600 µL of GP2 and centrifuge at 13,000 rpm for 1 min.
  6. Repeat step 5.
  7. Remove filtrate and centrifuge at 13,000 rpm for 2 min.
  8. Set column into 1.5 mL tube and add 50 µL of GP6.
  9. Centrifuge at 13,000 rpm for 2 min.

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)

SequenceTemp. (°C)Time (sec)
194120
29810
36830sec / 1kbp
44Hold
Cycle: sequence2~3 × (25~45)

3STEP Cycle (Tm value ≤ 63C)

SequenceTemp. (°C)Time (sec)
194120
29810
3Tm30
46830sec / 1kbp
54Hold
Cycle: sequence2~4 × (25~45)

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
SequenceTemp. (°C)Time (sec)
19610
2505
360240
44Hold
Cycle: sequence2~4 × 25

Ethanol precipitation

Solution PCR product DW 3M NaOAc Glycogen 100% EtOH
Volume (µL) 10 10 2 1 50
  1. centrifuge at 15,000 rpm for 15 min at room temprature
  2. Remove supernatant ,add 100 µL of 70% EtOH and tap tubes by finger.
  3. centrifuge at 15,000 rpm for 10 min at room temprature
  4. Remove supernatant and air dry at room temperature, after that 10 µL of  DW is added and dissolve the precipitate.
  5. Electrophoresis
  6. Resuspend the pellet to HiDi formamide and remove to 96-well plate.
  7. Set the plate and start electrophoresis.

Streaking (Single colony isolation)

  1. Pick the colony with an inoculating loop from the agar plate.
  2. Drag the loop across on a new agar plate.
  3. Re-sterilise the loop and drag it across again.
  4. Repeat method 3.

Colony PCR

Solution DNA Kapa-Taq (Taq polymerase) EX-F primer 10µM PS-R primer 10µM DW Total
Volume (µL) 4 10 0.8 0.8 8.4 20
SequenceTemp. (°C)Time (sec)
195120
29530
368.930
47260
572120
64Hold
cycles: sequence2~4 × 30~45

β-Galactosidase assay

(OZBIOSCHIENCES CPRG β-GAlactosidase Assay Kit : improved version)
  1. Liquid culture transfected cells with a plasmid coding LacZ gene for 24 hrs in 2mL LB.
  2. Centrifuge 100 µL of culture at 1000 G for 3 min.
  3. Remove the supernatant and suspend the pellet in 50 µL of 5x Lysis Buffer.
  4. Incubate the lysate for 15 min at room temperature by vortexing it several times to ensure complete lysis.
  5. Add 50 µL of Standard Dilution Buffer to 13-well(changeable) of a 96-well plate, and make a standard curve.
β-Gal Standard (milliunits) Standard Dilution Buffer Volume β-Gal Standard Volume
20 999 µL 1 µL of β-gal standard stock
10 100 µL 100 µL of 20 mu β-gal standard
5 100 µL 100 µL of 10 mu β-gal standard
2.5 100 µL 100 µL of 5 mu β-gal standard
1.25 100 µL 100 µL of 2.5 mu β-gal standard
0.625 100 µL 100 µL of 1.25 mu β-gal standard
0.3125 100 µL 100 µL of 0.625 mu β-gal standard
0.15625 100 µL 100 µL of 0.3125 mu β-gal standard
0.078125 100 µL 100 µL of 0.15625 mu β-gal standard
0.0390625 100 µL 100 µL of 0.078125 mu β-gal standard
0.01953125 100 µL 100 µL of 0.0390625 mu β-gal standard
0.009765625 100 µL 100 µL of 0.01953125 mu β-gal standard
0 100 µL
  1. Add 50 µL of each sample to new well.
  2. Add 50 µL of 1x CPRG Substrate Solution to each well and incubate the plate at room temperature for about 2 hrs.
  3. Add 100 µL of Stop Buffer to each well.
  4. Monitor the color development(Measure the absorbance of each sample) at 570-595 nm.
  5. Calculate the expression levels(the activity) of lacZ based on a standard curve

Golden Gate Assembly

PCR insert DNA with BsaI-adding primer (please refer Primer Designer for Maestro), purify the PCRed DNA and measure their concentrations.

Then, mix the following reagents in 0.2 mL PCR tube. Use BsaI / CutSmartTM, T4 DNA Ligase and its buffer (New England Biolabs Ltd). For vector DNA, use BBa_K1084501 etc..

SolutionBsaI10× CutSmart bufferT4 Ligase10× T4 buffervector DNAeach insert DNADWTotal
Volume1 µL1.5 µL1 µL1.5 µL100 ngequimolar with vector DNAfor messing up15 µL

Used PCR machine to digest and ligate in one-pot.

SequenceTemp. (°C)Time (min)
1373
2164
3505
4805
54Hold
cycles: sequence1~2 × 25