Team:Freiburg/Notebook/lab multiple targeting

From 2013.igem.org

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<div id="h1">
<div id="h1">
-
Multiple targeting Notebook
+
crRNA - Multiple targeting  
 +
</div>
 +
 
 +
<h3>12.08.13</h3>
 +
  <h5>Digest of reporter plasmids</h5>
 +
<div id="floatleft">
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> type </th>
 +
</tr>
 +
<tr>
 +
<td> 3 µg </td>
 +
<td> DNA (pKM602, pKM608, pKM611) </td>
 +
</tr>
 +
<tr>
 +
<td> 5 </td>
 +
<td> NEB-Buffer 4 </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Nhe I HF </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Ssp I HF </td>
 +
</tr>
 +
<tr>
 +
<td> 0,5 </td>
 +
<td> BSA </td>
 +
</tr>
 +
<tr>
 +
<td> Add to 50&micro;l </td>
 +
<td> H<sub>2</sub>O </td>
 +
</tr>
 +
</table>
 +
 
 +
<ul>
 +
<li> Temp.: 37&deg;C </li>
 +
<li> Incubation time: 2h </li>
 +
</ul>
 +
</div>
 +
 
 +
 
 +
 
 +
<div id="floatleft">
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> type </th>
 +
</tr>
 +
<tr>
 +
<td> 3 µg </td>
 +
<td> DNA (pIG7001b, pIG7002b, pIG7004b) </td>
 +
</tr>
 +
<tr>
 +
<td> 5 </td>
 +
<td> Promega MC Buffer </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Promega Nhe I </td>
 +
</tr>
 +
<tr>
 +
<td> 2 </td>
 +
<td> Promega Nru I </td>
 +
</tr>
 +
<tr>
 +
<td> 0.5 </td>
 +
<td> BSA </td>
 +
</tr>
 +
<tr>
 +
<td> Add to 50&micro;l </td>
 +
<td> H<sub>2</sub>O </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div id="floatright">
 +
<ul>
 +
<li> Temp.: 37&deg;C </li>
 +
<li> Incubation time: o/n </li>
 +
</ul>
 +
</div>
 +
 
 +
<h3>13.08.13</h3>
 +
  <h5>Gel run</h5>
 +
<p>A) Digest of pIG700..</p>
 +
<div id="floathleft">
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src="https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1376480056&w=500&h=376&media=wiki:laborbuecher:michael:13.08.13_digest_of_pig700x.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> From left to right: Marker (1 kb Roth); pIG7001; pIG7002; pIG7004 (50 & 4 µl); pIG7004 undigested (4 µl) </td>
 +
</tr>
 +
</table>
 +
 +
</div>
 +
 
 +
<div id="floathright">
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src="https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1376480056&w=500&h=376&media=wiki:laborbuecher:michael:13.08.13_digest_of_pig700x_cut.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> From left to right: Marker (1 kb Roth); pIG7001; pIG7002; pIG7004 (50 & 4 µl); pIG7004 undigested (4 µl) </td>
 +
</tr>
 +
</table>
 +
 +
</div>
 +
 
 +
<p>B) Digest of pKM60..</p>
 +
<div id="floathleft">
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src="https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1376480056&w=500&h=376&media=wiki:laborbuecher:michael:13.08.13_digest_of_pkm60x.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> From left to right: Marker (2 log NEB); pKM602; pKM608; pKM611 (50 & 4 µl) </td>
 +
</tr>
 +
</table>
 +
 
 +
</div>
 +
 
 +
<div id="floathleft">
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src="https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1376480056&w=500&h=376&media=wiki:laborbuecher:michael:13.08.13_digest_of_pkm60x_cut.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> From left to right: Marker (2 log NEB); pKM602; pKM608; pKM611 (50 & 4 µl) </td>
 +
</tr>
 +
</table>
 +
 +
</div>
 +
 
 +
<p>All bands are at the expected sizes.</p>
 +
 
 +
  <h5>Gel extraction</h5>
 +
<p>DNA were purified using <i>High Pure Plasmid Isolation Kit</i> of Roche.</p>
 +
<p>Changes to the protocol:
 +
<ul>
 +
<li>incubation at 56 °C for 10 min for gel dissolving</li>
 +
<li>elution with dH<sub>2</sub>O (incubation at 50 °C for 4 min before centrifugation)</li>
 +
</ul>
 +
<p>Yield: 10 ng/µl (pIG700..); 2 ng/µl (pKM60..)</p>
 +
 
 +
  <h5>Recombination of cutted parts</h5>
 +
 
 +
<div class="floatleft">
 +
 
 +
<table class="tabelle">
 +
 
 +
<tr> <th> ingredient </th> <th align="right"> amount </th> </tr>
 +
 
 +
<tr> <td> pIG7001/2 </td> <td align="right"> 2.5 µl </td> </tr>
 +
 
 +
<tr> <td> pKM602/11 </td> <td align="right"> 2 µl </td> </tr>
 +
 
 +
<tr> <td> T4-Ligase </td> <td align="right"> 1 µl </td> </tr>
 +
 
 +
<tr> <td> T4-Ligase buffer </td> <td align="right"> 2 µl </td> </tr>
 +
 
 +
<tr> <td> dH<sub>2</sub>O </td> <td align="right"> up to 20 µl </td> </tr>
 +
 
 +
</table>
 +
 
 +
Incubation at 22 °C for 1 h.
 +
 
 +
</div>
 +
 
 +
<div class="floatright">
 +
 
 +
<table class="tabelle">
 +
 
 +
<tr> <th> ingredient </th> <th align="right"> amount </th> </tr>
 +
 
 +
<tr> <td> pIG7004 </td> <td align="right"> 4.3 µl </td> </tr>
 +
 
 +
<tr> <td> pKM608 </td> <td align="right"> 1.2 µl </td> </tr>
 +
 
 +
<tr> <td> T4-Ligase </td> <td align="right"> 1 µl </td> </tr>
 +
 
 +
<tr> <td> T4-Ligase buffer </td> <td align="right"> 2 µl </td> </tr>
 +
 
 +
<tr> <td> dH<sub>2</sub>O </td> <td align="right"> up to 20 µl </td> </tr>
 +
 
 +
</table>
 +
 
 +
Incubation at 22 °C for 1 h.
 +
 
 +
</div>
 +
 
 +
  <h5>Trafo</h5>
 +
<div class="floatleft">
 +
<ol>
 +
<li>4 µl of plasmid were added to 25 µl of chemically competent <i>E. coli</i> cells</li>
 +
<li>incubation for 10 min on ice</li>
 +
<li>heatshock (42 °C for 45 s)</li>
 +
<li>incubation for 2 min on ice</li>
 +
<li>addition of 300 µl LB medium</li>
 +
<li>incubation for 1 h at 37 °C (shaking)</li>
 +
<li>distribution of 300 µl on LB plates with kanamycin</li>
 +
<li>incubation over night at 37 °C </li>
 +
</ol>
 +
</div>
 +
 
 +
<h3>14.08.13</h3>
 +
  <h5>Picking of clones</h5>
 +
<p>From each Ligation 7 clones were picked and spread on 1/4 plates.</p>
 +
 
 +
<h3>15.08.13</h3>
 +
  <h5>Miniprep</h5>
 +
<p>DNA was purified using <i>High Pure Plasmid Isolation Kit</i> of Roche.</p>
 +
<p>Yield: ~ 220 ng/µl</p>
 +
 
 +
  <h5>Test digest</h5>
 +
<div class="floatleft">
 +
<table class="tabelle">
 +
<tr> <th> ingredient </th> <th align="right"> volume </th> </tr>
 +
<tr> <td> plasmids (220 ng/µl)  </td> <td align="right"> 1.2 µl </td> </tr>
 +
<tr> <td> EcoRV-HF </td> <td align="right"> 0.5 µl </td> </tr>
 +
<tr> <td> NEB buffer 4 </td> <td align="right"> 1 µl </td> </tr>
 +
<tr> <td> dH<sub>2</sub>O </td> <td align="right"> up to 10 µl </td> </tr>
 +
</table>
 +
Incabation at 37 °C for 2 h.
 +
</div>
 +
 
 +
  <h5>Gel run</h5>
 +
<div id="floathleft">
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src="https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1376594355&w=500&h=376&media=15.08.13_test_digest_of_pig700x.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> From left to right: Marker (1 kb Roth); pIG7005 (3x); pIG7006 (3x); pIG7007 (3x) </td>
 +
</tr>
 +
</table>
 +
  <p>pIG7005_2, all clones of pIG7006 and pIG7007_1 & 3 showed the expected bands. pIG7005_2, pIG7006_1 and pIG7007_3 were send in for sequencing.</p>
 +
</div>
 +
 
 +
<h3>16.08.13</h3>
 +
  <h5>Midiprep</h5>
 +
<p>Though the sequencing did not have a result, plasmids were amplified and midiprepped by <i>Pure Yield Plasmid Midiprep System</i> from Promega because of the results of the test digest.</p>
 +
 
 +
  <h5>Seeding of cells</h5>
 +
<p>A 24 well plate with cover slips was filled with 50,000 cells per well.</p>
 +
 
 +
<h3>17.08.13</h3>
 +
  <h5>Transfection</h5>
 +
<div class="floatleft">
 +
Protocol:
 +
<ol>
 +
<li>40 µl Opti-MEM + 1.5 µl PEI-solution were mixed in a 1.5 ml Eppi.</li>
 +
<li>0.5 µg of the DNA of interest were prepaired in another Eppi .</li>
 +
<li>Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT</li>
 +
<li>Solution was spread drop-wise to the cells in the dish</li>
 +
</div>
 +
Transfection scheme:
 +
<div id="floathleft">
 +
<img class="gelpic" src="https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1376814433&w=500&h=283&media=wiki:laborbuecher:michael:transfection_multiple_targets_i.png">
 +
</div>
 +
 
 +
<h3>18.08.13</h3>
 +
  <h5>Fixation</h5>
 +
<p>Cells were washed with 500 µl PBS per well and fixated with 200 µl PFA per well (incubation on ice for 45 min).</p>
 +
<p>Cover slips were washed in dH<sub>2</sub>O and mounted on a drop of Mowiol with DABCO on a object slide.</p>
 +
 
 +
<h3>18.08.13</h3>
 +
  <h5>Flourescence microscopy</h5>
 +
<p>GFP and mCherry were expressed in all cells, only in the cells with TetR-VP16 there was a stronger signal. BFP was not detectable because of the microscope.</p>
 +
 
 +
<h3>21.08.13</h3>
 +
 
 +
  <h5>Seeding of cells</h5>
 +
<p>A 24 well plate with cover slips was filled with 50,000 CHO cells per well.</p>
 +
 
 +
<h3>22.08.13</h3>
 +
  <h5>Transfection</h5>
 +
<div class="floatleft">
 +
Protocol:
 +
<ol>
 +
<li>40 µl Opti-MEM + 1.5 µl PEI-solution were mixed in a 1.5 ml Eppi.</li>
 +
<li>0.75 µg of the DNA of interest were prepaired in another Eppi .</li>
 +
<li>Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT</li>
 +
<li>Solution was spread drop-wise to the cells in the dish</li>
</div>
</div>
 +
  <h5>Medium change</h5>
 +
Medium was changed after 3 h.
 +
<h3>23.08.13</h3>
 +
  <h5>Fixation</h5>
 +
<p>Cells were washed with 500 µl PBS per well and fixated with 200 µl PFA per well (incubation on ice for 45 min).</p>
 +
<p>Cover slips were washed in dH<sub>2</sub>O and mounted on a drop of Mowiol with DABCO on a object slide.</p>

Revision as of 23:51, 12 September 2013

crRNA - Multiple targeting

12.08.13

Digest of reporter plasmids
µl type
3 µg DNA (pKM602, pKM608, pKM611)
5 NEB-Buffer 4
1 Nhe I HF
1 Ssp I HF
0,5 BSA
Add to 50µl H2O
  • Temp.: 37°C
  • Incubation time: 2h
µl type
3 µg DNA (pIG7001b, pIG7002b, pIG7004b)
5 Promega MC Buffer
1 Promega Nhe I
2 Promega Nru I
0.5 BSA
Add to 50µl H2O
  • Temp.: 37°C
  • Incubation time: o/n

13.08.13

Gel run

A) Digest of pIG700..

From left to right: Marker (1 kb Roth); pIG7001; pIG7002; pIG7004 (50 & 4 µl); pIG7004 undigested (4 µl)
From left to right: Marker (1 kb Roth); pIG7001; pIG7002; pIG7004 (50 & 4 µl); pIG7004 undigested (4 µl)

B) Digest of pKM60..

From left to right: Marker (2 log NEB); pKM602; pKM608; pKM611 (50 & 4 µl)
From left to right: Marker (2 log NEB); pKM602; pKM608; pKM611 (50 & 4 µl)

All bands are at the expected sizes.

Gel extraction

DNA were purified using High Pure Plasmid Isolation Kit of Roche.

Changes to the protocol:

  • incubation at 56 °C for 10 min for gel dissolving
  • elution with dH2O (incubation at 50 °C for 4 min before centrifugation)

Yield: 10 ng/µl (pIG700..); 2 ng/µl (pKM60..)

Recombination of cutted parts
ingredient amount
pIG7001/2 2.5 µl
pKM602/11 2 µl
T4-Ligase 1 µl
T4-Ligase buffer 2 µl
dH2O up to 20 µl
Incubation at 22 °C for 1 h.
ingredient amount
pIG7004 4.3 µl
pKM608 1.2 µl
T4-Ligase 1 µl
T4-Ligase buffer 2 µl
dH2O up to 20 µl
Incubation at 22 °C for 1 h.
Trafo
  1. 4 µl of plasmid were added to 25 µl of chemically competent E. coli cells
  2. incubation for 10 min on ice
  3. heatshock (42 °C for 45 s)
  4. incubation for 2 min on ice
  5. addition of 300 µl LB medium
  6. incubation for 1 h at 37 °C (shaking)
  7. distribution of 300 µl on LB plates with kanamycin
  8. incubation over night at 37 °C

14.08.13

Picking of clones

From each Ligation 7 clones were picked and spread on 1/4 plates.

15.08.13

Miniprep

DNA was purified using High Pure Plasmid Isolation Kit of Roche.

Yield: ~ 220 ng/µl

Test digest
ingredient volume
plasmids (220 ng/µl) 1.2 µl
EcoRV-HF 0.5 µl
NEB buffer 4 1 µl
dH2O up to 10 µl
Incabation at 37 °C for 2 h.
Gel run
From left to right: Marker (1 kb Roth); pIG7005 (3x); pIG7006 (3x); pIG7007 (3x)

pIG7005_2, all clones of pIG7006 and pIG7007_1 & 3 showed the expected bands. pIG7005_2, pIG7006_1 and pIG7007_3 were send in for sequencing.

16.08.13

Midiprep

Though the sequencing did not have a result, plasmids were amplified and midiprepped by Pure Yield Plasmid Midiprep System from Promega because of the results of the test digest.

Seeding of cells

A 24 well plate with cover slips was filled with 50,000 cells per well.

17.08.13

Transfection
Protocol:
  1. 40 µl Opti-MEM + 1.5 µl PEI-solution were mixed in a 1.5 ml Eppi.
  2. 0.5 µg of the DNA of interest were prepaired in another Eppi .
  3. Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
  4. Solution was spread drop-wise to the cells in the dish
Transfection scheme:

18.08.13

Fixation

Cells were washed with 500 µl PBS per well and fixated with 200 µl PFA per well (incubation on ice for 45 min).

Cover slips were washed in dH2O and mounted on a drop of Mowiol with DABCO on a object slide.

18.08.13

Flourescence microscopy

GFP and mCherry were expressed in all cells, only in the cells with TetR-VP16 there was a stronger signal. BFP was not detectable because of the microscope.

21.08.13

Seeding of cells

A 24 well plate with cover slips was filled with 50,000 CHO cells per well.

22.08.13

Transfection
Protocol:
  1. 40 µl Opti-MEM + 1.5 µl PEI-solution were mixed in a 1.5 ml Eppi.
  2. 0.75 µg of the DNA of interest were prepaired in another Eppi .
  3. Addition of the DNA to former Eppi, vortexing for 10 s and incubation for 15 min at RT
  4. Solution was spread drop-wise to the cells in the dish
Medium change
Medium was changed after 3 h.

23.08.13

Fixation

Cells were washed with 500 µl PBS per well and fixated with 200 µl PFA per well (incubation on ice for 45 min).

Cover slips were washed in dH2O and mounted on a drop of Mowiol with DABCO on a object slide.

Retrieved from "http://2013.igem.org/Team:Freiburg/Notebook/lab_multiple_targeting"