Team:Freiburg/Notebook/lab light

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<div id="h1">
<div id="h1">
-
Light Notebook
+
Induction - Light
</div>
</div>
 +
 +
<h2>29.07.2013</h2>
 +
<p> Plasmids that we need as templates were transformed. </p>
 +
<h2>30.07.2013</h2>
 +
<h3>Primers</h3>
 +
<p>Primers arrived today. Primers were diluted 1:10.</p>
 +
<h3>Minipreps</h3>
 +
<p>Transformed bacteria from yesterday with our template plasmids were minipreped.</p>
 +
<ul>
 +
<li> pKM018: 83 ng/µl</li>
 +
<li> pKM220: 216 ng/µl</li>
 +
<li> pKM115: 177 ng/µl</li>
 +
<li> pKM006: 118 ng/µl</li>
 +
<li> pSAM200: 213 ng/µl</li>
 +
<li> pSW42: 134 ng/µl</li>
 +
<li> pSW45: 243 ng/µl</li>
 +
<li>pIG2004: 212 ng/µl</li>
 +
<li>T405: 213 ng/µl</li>
 +
</ul>
 +
<h3> Oligo Annealing of tetO crRNA </h3>
 +
<ul>
 +
<li> Oligos tetO13 1. CC and tetO13 2. CC were annealed
 +
<li> Therefore 20 µM diluted Primes were heated on 98° C for 4 minutes and then cooled down.
 +
</ul>
 +
 +
<h3> PCR list, program and composition</h3>
 +
 +
<ul>
 +
<li> aIG4102: CMV Promotor from template T405 - Primers oIG4100F+R - 589 bp </li>
 +
<li> aIG4103: Cas9 from template pIG2004 - Primers oIG4101F+R - 4170 bp </li>
 +
<li> aIG4104: PIF and backbone from template pKM022- Primers oIG4102F+R - 3287 bp</li>
 +
<li> aIG4105: KRAB from template pIG2004 - Primers oIG4103F+R - 390 bp </li>
 +
<li> aIG4106: PhyB and backbone from template pKM018 - Primers oIG4104F+R - 4922 bp </li>
 +
<li> aIG4107: UVR8 and backbone from template pKM168 - Primers oIG4105F+R - 3921 bp </li>
 +
<li> aIG4108: COP1 and backbone frome template pKM115 - Primers oIG4106F+R - 3972 bp </li>
 +
<li> aIG4109: CRY2 from template pSW42 - Primers oIG4107F+R - 1591 bp </li>
 +
<li> aIG4110: Backbone from template pKM018 - Primers oIG4108F+R - 2924 bp </li>
 +
<li> aIG4111: Backbone with VP16 from template pKM018 - Primers oIG4109F+R - 3282 bp </li>
 +
<li> aIG4112: CIB from template pSW45 - Primers oIG4110F+R - 593 bp </li>
 +
<li> aIG4113: Backbone from template pKM018 - Primers oIG4111F+R - 2881 bp </li>
 +
<li> aIG4114: CIB from template pSW45 - Primers !! oIG4110F+oIG4112R - 638 bp </li>
 +
</ul>
 +
 +
<div id="${1:floatleft}">
 +
<table class="${2:tabelle}">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> Substance </th>
 +
</tr>
 +
<tr>
 +
<td> 10 </td>
 +
<td> Q5-HF Reaction Buffer </td>
 +
</tr>
 +
<tr>
 +
<td> 200 ng </td>
 +
<td> Template </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Primer forward </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Primer reverse </td>
 +
</tr>
 +
<tr>
 +
<td> 4 </td>
 +
<td> dNTPs </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> DMSO </td>
 +
</tr>
 +
<tr>
 +
<td> 0,5 </td>
 +
<td> Q5-HF Polymerase </td>
 +
</tr>
 +
<tr>
 +
<td> Add to 50 </td>
 +
<td> H2O </td>
 +
</tr>
 +
</table>
 +
</div>
 +
 +
<div id="${1:floatleft}">
 +
<table class="${2:tabelle}">
 +
<tr>
 +
<th> Temperature [° C] </th>
 +
<th> Time </th>
 +
</tr>
 +
<tr>
 +
<td> 98 </td>
 +
<td> 5 Min </td>
 +
</tr>
 +
<tr>
 +
<td> 98 </td>
 +
<td> 30s </td>
 +
</tr>
 +
<tr>
 +
<td> 60 (or optimized) </td>
 +
<td> 30 </td>
 +
</tr>
 +
<tr>
 +
<td> 72 </td>
 +
<td> 30-40/kb </td>
 +
</tr>
 +
<tr>
 +
<td> 72 </td>
 +
<td> 10 Min </td>
 +
</tr>
 +
<tr>
 +
<td> 4 </td>
 +
<td> hold </td>
 +
</table>
 +
</div>
 +
<div id="${18:floatright}">
 +
<ul>
 +
<li> 18 cycles </li>
 +
</ul>
 +
</div>
 +
<h3> PCR results </h3>
 +
<ul>
 +
<li> aIG4102: 60° C - WORKED - GelExtraction: 87,9ng/ul </li>
 +
<li> aIG4103: 60° C - 1 of 4 WORKED, Annealing Temperature lower? - GelExtraction: 50,5ng/ul <b>Try 56°!</b> </li>
 +
<li> aIG4104: 60° C - DIDN'T WORK!!! Try with 58° - <b>Was done, has to be tested on gel.</b></li>
 +
<li> aIG4105: 60° C - WORKED - GelExtraction: 70,1ng/ul I and 57,7ng/ul II </li>
 +
<li> aIG4106: 60° C - <b>Was done, has to be tested on gel.</b></li>
 +
<li> aIG4107: 60° C - ONLY SLIGHT BAND. TRY AGAIN! </li>
 +
<li> aIG4108: 60° C - WRONG AMPLIFICATED BAND. <b>Try again with different temperature! </b></li>
 +
<li> aIG4109: 60° C - WORKED! - GelExtraction: 176,4ng/ul </li>
 +
<li> aIG4110: 60° C - <b>Was done, has to be tested on gel.</b></li>
 +
<li> aIG4111: 60° C - WORKED! - GelExtraction: 84,4ng/ul </li>
 +
<li> aIG4112: 60° C - <b>Wasn't done yet.</b></li>
 +
<li> aIG4113: 60° C - <b>Was done, has to be tested on gel.</b></li>
 +
<li> aIG4114: 60° C - <b>Wan't done yet.</b></li>
 +
</ul>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src="https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375227099&w=500&h=376&media=wiki:laborbuecher:07-30_aig4102_und_aig4105.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log Marker</p> <p>aIG4102 (first 4) <b>worked</b>, 589 bp</p> <p>aIG4105 (rest) worked, 390 bp</p> </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src="https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375227103&w=500&h=376&media=wiki:laborbuecher:07-30_aig4103.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>Roth Marker</p> <p><b>aIG4103</b> <b>only one PCR worked</b>, 4170 bp</p></td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src="https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375227105&w=500&h=376&media=wiki:laborbuecher:07-30_aig4104_links_und_aig4111_rechts.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>Roth Marker</p> <p><b>aIG4104</b> didn't work</p> <p><b>aIG4111 worked</b>, 3282 bp</p></td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src="https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375227110&w=500&h=376&media=wiki:laborbuecher:07-30_aig4109_und_aig4107_und_aig4108.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log Marker</p> <p><b>aIG4109</b> (first two bands) <b>worked</b>, 1591 bp</p> <p><b>aIG4107</b> (next two bands) didn't work, should have been 3921 bp</p> <p><b>aIG4108</b> (one band) didnt work. Wrong band was amplified, should have been 3972 bp - another band of aIG4108 can be tested on a gel, didn't have space on this one.</p></td>
 +
</tr>
 +
</table>
 +
</div>
 +
<h2>31.07.13</h2>
 +
<h3>PCR results</h3>
 +
<ul>
 +
<li> aIG4103: 54°-64° C - Everything WORKED! </li>
 +
<li> aIG4104: 58° C - Gave three bands, upper band is supposed to be right. Was confirmed on a second gel with charging the gel only with a small amount of DNA.</b></li>
 +
<li> aIG4106: 60° C - Didn't work. <b> Try temperature gradient. </b></li>
 +
<li> aIG4107: 54°-64° C - Everything Worked! But not very strong bands. </li>
 +
<li> aIG4108: 54°-64° C - Didn't Work. <b> Try seperate PCRs. </b> </b></li>
 +
<li> aIG4110: 60° C - Didn't work. Tried temperature gradient 54°-59° C - Worked with 55° and better with every additional ° C.</b></li>
 +
<li> aIG4112: 60° C - Worked!</li>
 +
<li> aIG4113: 60° C - Worked!</b></li>
 +
<li> aIG4114: 60° C - Worked!</li>
 +
</ul>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src="https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375395270&w=500&h=376&media=wiki:laborbuecher:07-31_aig4103_2_-_aig4107_2_-_aig4106_5-10_-_aig4108_unten.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log marker</p> <p> UPPER SIDE </p> <p>aIG4103 (2 bands) - worked!</p> <p> aIG4107 (2 bands) - worked! </p> <p> aIG4106 (4 bands) - didn't work with 60° C, bands are too low! <p> <p> LOWER SIDE </p> aIG4108 temperature gradient 54°-64° C - didn't work!</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src="https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375395259&w=500&h=376&media=wiki:laborbuecher:07-31_aig4103_temperaturgradient_54-64.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log marker</p> <p> aIG4103 temperature gradient 54°-64° C - everything worked! </p> </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src="https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375395264&w=500&h=376&media=wiki:laborbuecher:07-31_aig4105_-_aig4104_-_aig4110.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log marker</p> <p> aIG4105 (2bands) - worked! </p> <p> aIG4104 (2 bands) - upper band is considered to be the right band! - Worked! </p> <p> aIG4110 - didn't work with 60° C.</p> </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src="https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375395279&w=500&h=376&media=wiki:laborbuecher:07-31_aig4107_temperaturgradient.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log marker</p> <p>aIG4107 temperature gradient 54°-64° C - worked, but only slight bands! </p> </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375395289&w=500&h=376&media=wiki:laborbuecher:07-31_aig4108_-_aig4113_-_aig4110.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log marker</p> <p> aIG4108 (2bands) - didn't work with 60° C! </p> <p> aIG4113 (2 bands) - Worked! </p> <p> aIG4110 - didn't work with 60° C.</p> </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375395294&w=500&h=376&media=wiki:laborbuecher:07-31_aig4110_1_und_aig4112_2_und_aig4114_2_.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log marker</p> <p> aIG4110 (1 band) last column of temperature gradient (59° C) - worked! </p> <p> aIG4112 (2 bands) - Worked! </p> <p> aIG4114 - worked!.</p> </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375395294&w=500&h=376&media=wiki:laborbuecher:07-31_aig4110.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log marker</p> <p> aIG4110 temperature gradient (54°-59° C) - worked from 55° C upwards! </p></td>
 +
</tr>
 +
</table>
 +
</div>
 +
<h2>01.08.13</h2>
 +
<h3>PCR results</h3>
 +
<ul>
 +
<li> aIG4104: Was gelexed and size was confirmed on a gel with a smaller amount of DNA. - Worked!
 +
<li> aIG4106: Temperature gradient 54° - 59° C and excess of reverse primer oIG4104R - Worked! </b></li>
 +
<li> aIG4108: Seperate PCRs were tried out. Therefore the PCR was performed with template and at a time only one primer. After these PCRs (1x with forward Primer (58° C) and 1x with reverse Primer (62° C)) both columns were mixed and another PCR was performed. - Worked, but two bands appeared, upper band is considered to be the right fragment. </b></li>
 +
</ul>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375396754&w=500&h=376&media=wiki:laborbuecher:08-01_aig4106_aig4104_1_.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log marker</p> <p> aIG4106 temperature gradient (54°-59° C) - worked! </p></td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375396754&w=500&h=376&media=wiki:laborbuecher:08-01_aig4108.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log marker</p> <p> aIG4108 seperate PCRs - worked, but other amplified products can be seen, too! </p></td>
 +
</tr>
 +
</table>
 +
</div>
 +
<h3>Gibsons</h3>
 +
                        <tr>  <p>  Fragments are mixed together to a total volume of 5µl and added to 15µl Gibson Master- Mix.</p>
 +
                                        </tr>
 +
                                        <tr>
 +
                                                <p> <td> pIG4101: aIG4104- 0,83µl; aIG4103- 3,96µl; H2O- 0,21µl </td> </p> <p>
 +
 +
                                                <td> pIG4103: aiG4107- 2,12µl; aiG4103- 2,88µl </td> </p> <p>
 +
                                                <td> pIG4105: aIG4110- 1,11µl; aIG4103- 3,51µl </td> </p> <p>
 +
                                                <td> pIG4106: aIG4111- 0,5µl;  aIG4112- 0,3µl, H2O- 4,2ul </td> </p> <p>
 +
                                                <td> pIG4107: aIG4113- 0,4µl;  aIG4105- 0,35µl; aIG4114- 0,35µl; H2O- 3,9µl </td>
 +
<p> Gibson Mixes were transformed. <p>
 +
 +
<h3>Ligation of pIG4100</h3>
 +
<h4>Digest for pIG4100</h4>
 +
<div id="floatleft">
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> type </th>
 +
</tr>
 +
<tr>
 +
<td> 10 </td>
 +
<td> DNA (pKM006 or oIG4102) ~ 1000 ng </td>
 +
</tr>
 +
<tr>
 +
<td> 4 </td>
 +
<td> Cut Smart Buffer </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> EcoRI HF </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Nhe1 HF </td>
 +
</tr>
 +
<tr>
 +
<td> Add to 40&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: 1,5h </li>
 +
</ul>
 +
</div>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375397380&w=500&h=376&media=wiki:laborbuecher:08-01_verdau_licht.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log marker</p> <p> Digested aIG4102 with 601 bp </p> <p> Digested pKM006 with 6393 bp and 126 bp. </p> <p> Both were gelexed: aIG4102 19 ng/µl and pKM006 40 ng/µl</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<h4>Ligation and Transformation of approach pIG4100</h4>
 +
<div id="floatleft">
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> type </th>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> T4 Ligase </td>
 +
</tr>
 +
<tr>
 +
<td> 2 </td>
 +
<td> T4 Ligase buffer </td>
 +
</tr>
 +
<tr>
 +
<td> 3 fold molar amount of insert </td>
 +
</tr>
 +
<tr>
 +
<td> 2 </td>
 +
<td> Backbone pKM006 </td>
 +
</tr>
 +
<tr>
 +
<td> 1,3 </td>
 +
<td> Insert aIG4102 </td>
 +
</tr>
 +
<tr>
 +
<td> 13,7 </td>
 +
<td> H<sub>2</sub>O </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div id="floatright">
 +
<ul>
 +
<li> Temp.: RT </li>
 +
<li> Incubation time: 15 minutes </li>
 +
</ul>
 +
</div>
 +
 +
<p> After the ligation, 2,5 µl if this approach were transformed into E.coli and scratched out.</p>
 +
 +
 +
<h2>02.08.13</h2>
 +
<h3>Gibson & Ligation results</h3>
 +
<p> Colonies could be seen on every plate except for one of the two ligation plates. Clones were picked and scratched out -> 51 Minis </p>
 +
 +
<h3> Gibson </h3>
 +
<p> <td> pIG4102: aIG4105- 0,4µl; aIG4106- 2,00µl; H2O- 2,6 µl </td> </p> <p>
 +
 +
                    <td> pIG4104: aiG4108- 3,2µl; aiG4105- 0,5µl; H2O- 1,3µl </td> </p>
 +
                      <p> Gibson Mixes were transformed. <p>
 +
 +
<h2>03.08.13</h2>
 +
<h3> Gibson results </h3>
 +
<p> Colonies could be seen on one of two plates, each. Colonies were scratched out for mini preps. Therefore we had clones for every construct. </p>
 +
 +
<h3> Minipreps </h3>
 +
<p> 51 Minis - concentrations between 50 and 200 ng/µl. </p>
 +
 +
 +
<h2>04.08.13</h2>
 +
<h3> Minipreps </h3>
 +
<p> 15 Minis. </p>
 +
 +
 +
<h2>05.08.13</h2>
 +
<h3> Test digest </h3>
 +
<p> Today we made a test digest session of our 66 minis with following enzymes. </p>
 +
<div id="floatleft">
 +
<table class="tabelle">
 +
<tr>
 +
<th> Plasmid </th>
 +
<th> Enzymes </th>
 +
<th> Size </th>
 +
</tr>
 +
<tr>
 +
<td> pIG4100 </td>
 +
<td> EcoRI-HF & EcoRV-HF</td>
 +
<td> <b>1571 bp</b> (Insert CMV 600bp) & <b>5417 bp</b></td>
 +
</tr>
 +
<tr>
 +
<td> pIG4101 </td>
 +
<td> EcoRV-HF & NotI-HF</td>
 +
<td> <b>1952 bp</b> (half the Cas9) & <b>5456 bp</b></td>
 +
</tr>
 +
<tr>
 +
<td> pIG4102 </td>
 +
<td> EcoRI-HF & NotI-HF </td>
 +
<td> <b>1966 bp</b> (with PhyB) & <b>3274 bp</b> </td>
 +
</tr>
 +
<tr>
 +
<td> pIG4103 </td>
 +
<td> BamHI-HF & XhoI </td>
 +
<td> <b>5337 bp</b> (Cas9, UVR8) & <b>2682 bp</b> </td>
 +
</tr>
 +
<tr>
 +
<td> pIG4104 </td>
 +
<td> NotI-HF & BamHI-HF </td>
 +
<td> <b>1451 bp</b> (with COP1) & <b>2839 bp</b> </td>
 +
</tr>
 +
<tr>
 +
<td> pIG4105 </td>
 +
<td> EcoRI-HF & EcoRV-HF </td>
 +
<td> <b>3040 bp</b> (Half the Cas9 and half CRY2) & <b>5529 bp</b></td>
 +
</tr>
 +
<tr>
 +
<td> pIG4106 </td>
 +
<td> NotI-HF & XbaI </td>
 +
<td> <b>970 bp</b> (CIB, VP16) & <b>2833 bp</b></td>
 +
</tr>
 +
<tr>
 +
<td> pIG4107 </td>
 +
<td> PstI-HF & NotI-HF </td>
 +
<td> <b>976 bp</b> (CIB, KRAB) & <b>2821 bp</b></td>
 +
</tr>
 +
 +
 +
</table>
 +
</div>
 +
<div id="floatleft">
 +
<table class="tabelle">
 +
<tr>
 +
<th> &micro;l </th>
 +
<th> type </th>
 +
</tr>
 +
<tr>
 +
<td> ~ 100-200 ng </td>
 +
<td> DNA </td>
 +
</tr>
 +
<tr>
 +
<td> 1 </td>
 +
<td> Cut Smart buffer </td>
 +
</tr>
 +
<tr>
 +
<td> 0,5 </td>
 +
<td> Enzyme 1 </td>
 +
</tr>
 +
<tr>
 +
<td> 0,5 </td>
 +
<td> Enzyme 2 </td>
 +
</tr>
 +
<tr>
 +
<td> Add to 10&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: 2h </li>
 +
</ul>
 +
</div>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375832566&w=500&h=376&media=wiki:laborbuecher:test_digest_-_pig4100_oben_und_pig4101_unten.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log marker</p> <p> Upper bands: pIG4100, expected sizes: Plasmid 3 and 5 were send to GATC</p> <p> Bottom bands: pIG4101, expected sizes: Plasmid 4 and 7 were send to GATC </p> </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375832563&w=500&h=376&media=wiki:laborbuecher:test_digest_-_pig4102.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log marker</p> <p> pIG4102, expected sizes, Plasmid 1 and 9 were send to GATC.</p> </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375832562&w=500&h=376&media=wiki:laborbuecher:test_digest_-_oben_links_pig4104_und_oben_rechts_pig41005_und_unten_pig4103.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log marker</p> <p> Upper bands left side: pIG4104, expected sizes: Plasmid 1 and 4 were send to GATC</p> <p> Upper bands right side: pIG4105, expected sizes: Plasmid 1 and 6 were send to GATC </p> </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375832573&w=500&h=376&media=wiki:laborbuecher:test_digest_-_pig4106_oben_und_pig4107_unten.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log marker</p> <p> Upper bands: pIG4106, <br>not expected</b> sizes: Plasmid 3 was send to GATC</p> <p> Bottom bands: pIG4107, expected sizes: Plasmid 1 and 4 were send to GATC </p> </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<p> For the sequencing we used GATC Primers: CMV-F for pIG4100 and EBV-RP (binds in pSV40) for all other plasmids. </p>
 +
<h3> Insertion of crRNA into RNA Plasmid </h3>
 +
<ol>
 +
<li> First RNA Plasmid backbone was digested with BbsI for 3 hours. Enzyme was taken out of - 80° C.</li>
 +
<li> Then it was put on a gel and gelexed. Concentration: 50,5ng/ug. Backbone from Hormone- Group: 8,7ng/ul
 +
<li> Ligation: Digested backbone from Hormone- Induction and self- digested backbone was used. Molar ratio of                        backbone : insert - 1 : 3. </li>
 +
                        <li> Mix: Backbone; Insert, 1ul T4- ligase, 2ul T4-ligase-buffer, H2O up to a total volume of 20ul </li>
 +
                        <li> Incubated for 15min at RT </li>
 +
<li> Transformed and scratched out.
 +
</ol>
 +
 +
<h2>06.08</h2>
 +
<h3> Sequencing results</h3>
 +
<p> Sequencing results were <br>positive for pIG4101, pIG4102, pIG4103, pIG4105, pIG4106 and pIG4107</br> (sequenced with EBV-RP -> SV40 Terminator). pIG4100 didn't have the CMV promotor as an insert (-> new ligation) and pIG4104 didn't contain KRAB, although KRAB worked with pIG4102 (new Primers?)</p>
 +
<h3> Re-Trafos and inoculation for midi-preps</h3>
 +
<p> The positive Plasmids and already existing plasmids pKM018 and pKM115 were retransformed and put into 150 ml LB with 150 µl Ampicillin and incubated at 37° C over night. <p>
 +
<h3> New Primers for pIG4104 </h3>
 +
<p> New Primers were designed for pIG4104. </p>
 +
<h3> Ligation of pIG4100 </h3>
 +
<p> Ligation of pIG4100 was done again like on 31.07.13, only the ratio of backbone and insert was changed (3 µl backbone and 1,5 µl Insert). </p>
 +
<div>
 +
<table class="gelpic">
 +
<tr>
 +
<td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1375832565&w=500&h=376&media=wiki:laborbuecher:verdau_08-06.jpg"> </td>
 +
</tr>
 +
<tr>
 +
<td> <p>2-log marker</p> <p> Left band: Backbone, Right band: Inser</p> </td>
 +
</tr>
 +
</table>
 +
</div>
 +
<h3> Result of Ligation of RNA Plasmid </h3>
 +
<p> There were clones on every plate. They were scratched out for minipreps.</p>
 +
<h3> New sequencing </h3>
 +
<p> Plasmids were send to GATC with different primers. Cas9 was sequenced completely in pIG4101.7. Other pIG4104 were screened for insertion of KRAB.</p>
 +
 +
        <h2> 7.8.2013 </h2>
 +
        <h3> Midipreps of pKM017; pIG4101.7 (?), pIG4102.1, pIG4103, pKM115, pIG4105.5, pIG4106.3, pIG4107.4 </h3>
 +
              <p> All midipreps were successfull </p>
 +
              <p> Concentrations: </p>
 +
              <p> pKM018: 474 ng/ul </p>
 +
              <p> pIG4101: 485 ng/ul </p>
 +
              <p> pIG4102: 528 ng /ul </p>
 +
              <p> pIG4103: 521 ng/ul </p>
 +
              <p> pKM115: 517 ng/ul </p>
 +
              <p> pIG4105.1: 492 ng /ul </p>
 +
              <p> pIG4106: 567 ng/ul </p>
 +
              <p> pIG4107: 476 ng /ul </p>
 +
        <h3> Minipreps of RNA Plasmid pIG4201 and pIG4202</h3>
 +
        <p> Minis were send to GATC. 3 of ligation with digested Backbone from Michi and tetO1 and 3 with tetO2. And 3 of ligation with newly digested Backbone from Gabi and tetO1 and 3 with tetO2.</p>
 +
        <h3> Result of Ligation of pIG4100</h3>
 +
        <p> There were clones on every plate. They were scratched out for minis.</p>
 +
 +
      <h2> 8.8.2013 </h2>
 +
      <h3> Sequencing of RNA Plasmid </h3>
 +
      <p> 1 of the 6 pIG4201 and 4 of the 6 pIG4202 were positiv with inserted crRNA.</p>
 +
      <h3> Sequencing Order </h3>
 +
      <p> Sequencing of pIG4107_2 and pIG4107_3 with Primer EBV-RP </p>
 +
      <h3> Minis of pIG4100</h3>
 +
      <p> 12 Minis were prepped. </p>
 +
      <h3> Re-Trafo for inoculation for Midi Preps</h3>
 +
      <p> The following plasmids were retransformed: pIG1000, pIG4201, pIG4202 </p>
 +
 +
      <h2> 9.8.2013 </h2>
 +
      <h3> Midis of ... </h3>
 +
      <h3> Test digest of pIG4100 </h3>
 +
      <h2> 10.8.2013 </h2>
 +
      <h3> Seeding of CHO cells </h3>
 +
      <p> CHO- cells were seeded: 70.000 cells per well on 24 well- plates in HTS-medium </p>
 +
      <h2> 11.8.2013 </h2>
 +
      <h3> Transfection of CHO cells </h3>
 +
          <td>  <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?media=1.jpg> </td>
 +
          <td>  <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?media=2.jpg> </td>
 +
          <td>  <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1376472011&w=500&h=404&media=wiki:laborbuecher:licht20:11-08_1_.jpg </td>
 +
          <td>  <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1376472011&w=500&h=404&media=wiki:laborbuecher:licht20:11-08_2_.jpg </td>
 +
      <h4> Protocol for 24- well plates: </h4>
 +
          <p> 1. Mix 40 µl Opti-MEM + 1.5 µl PEI-solution in a 1.5 ml Eppi </p>
 +
          <p> 2.Prepare in another Eppi 0.5 µg of the DNA of interest  </p>
 +
          <p> 3.Add DNA to former Eppi, vortex thoroughly and incubate 15 min at RT  </p>
 +
          <p> 4.Resuspend solution gently and spread them drop-wise to the cells in the dish  </p>
 +
          <p> 5.Swing the dish in axis-form  </p>
 +
          <p> 6.Change of media 5h post transfection  </p>
 +
 +
          <h2> 12.8.2013 </h2>
 +
              <h3> PCB- treatment of CHO-cells and illumination </h3>
 +
                    <p> Cells are treated with HTS-medium containing 15uM of phycocyanobilin </p>
 +
                    <p> illumination started at 16.15. with 20uE light- quantity  </p>
 +
                    <p> plate 1 with activating red light (660nm)  </p>
 +
                    <p> plate 2 with inctivating far-red light (740nm), control  </p>
 +
 +
                <h3> construction of pIG4104 </h3>
 +
                    <p> pKM115 was digested with BamHI and BssHII for 4h. KRAB was amplified from pIG2004 with oIG4103FII and oIG4103RII  </p>
 +
                    <p> Subsequently the fragments were assembled via Gibson-cloning and transformed into E.coli </p>
 +
 +
          <h2> 12.8.2013 </h2>
 +
                <h3> Gibson results </h3>
 +
                    <p> Cell-colonies are picked and plated out anew </p>
 +
                    <p> A mistake in the cloning strategy was discovered, new primers are designed </p>
 +
 +
          <h2> 13.8.2013 </h2>
 +
                <h3> Gibson results </h3>
 +
                      <p> CHO- cells are illuminated </p>
 +
 +
          <h2> 14.8.2013 </h2>
 +
                <h3> Seeding of CHO-cells </h3>
 +
                      <p> CHO- cells were seeded: 70.000 cells per well on 24 well- plates in HTS-medium </p>
 +
                <h3> SEAP-measurements of illuminated cells </h3>
 +
                      <p> The results are all negative except for the positive control (-> see transfection Nr.6 of 11.8.2013) </p>
 +
                      <p> The interaction with the cri-RNA may be responsible. The experiment is repeated with new target- sites  </p>
 +
                     
 +
          <h2> 15.8.2013 </h2>
 +
                <h3> Transfection of CHO-cells for red light and UV tests </h3>
 +
                    <p> The wiki- transfection protocoll is used:  </p>
 +
                    <p> 1. Mix 40 µl Opti-MEM + 1.5 µl PEI-solution in a 1.5 ml Eppi </p>
 +
                    <p> 2.Prepare in another Eppi 0.5 µg of the DNA of interest  </p>
 +
                    <p> 3.Add DNA to former Eppi, vortex thoroughly and incubate 15 min at RT  </p>
 +
                    <p> 4.Resuspend solution gently and spread them drop-wise to the cells in the dish  </p>
 +
                    <p> 5.Swing the dish in axis-form  </p>
 +
                    <p> 6.Change of media 5h post transfection  </p>
 +
               
 +
                  <p> The following pattern is transfected in 24- well plates: </p>
 +
                    <td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?media=wiki:laborbuecher:licht20:15-08_uv.jpg> </td>
 +
                    <td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?media=wiki:laborbuecher:licht20:15-08_rot_660nm.jpg> </td>
 +
                    <td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?media=wiki:laborbuecher:licht20:15-08_rot_740nm.jpg> </td>
 +
                   
 +
            <h2> 16.8.2013 </h2>
 +
                <h3> UV-b and red-/ far red light illumination of transfected cells from 15.8.2013 </h3>
 +
                    <p> Cells for red- light illumination are illuminated with 20uE; 660nm or 720nm for 24h. Illumination is started at 14.20  </p>
 +
                    <p> Cells for UV-b light illumination are illiminated with 5uE; 311nm for 24h  </p>
 +
                   
 +
            <h2> 17.8.2013 </h2>
 +
                    <p> SEAP-measurements taken from illuminated- and untreated cells. </P>
 +
                    <p> the following protocoll was obeyed for the whole process </p>
 +
                            <li>200 µl of cell supernatant were transfered form the 24-well plate into one 96-well plate (round bottom)</li>
 +
                            <li>Incubation for 30 min at 65 °C.</li>
 +
                            <li>Centrifugation for 1 min at 1250 g.</li>
 +
                            <li>100 µl of SEAP buffer were filled in the wells of a 96 well plate (flat bottom).</li>
 +
                            <li>Addition of 80 µl supernatant from the round bottom plate to each well with SEAP buffer.</li>
 +
                            <li>Addition of 20 µl pNPP (substrate).</li>
 +
                            <li>96 plate was immediately placed in the blade reader.</li>
 +
                                                                                                </ul>
 +
                          <p>Spectroscopic measurement every minute for 120 times (2h); wavelenght 405 nm.</p>
 +
                         
 +
<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378423063&w=500&h=325&media=wiki:laborbuecher:licht20:rl_17-08.png> </td>
 +
<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378423064&w=500&h=340&media=wiki:laborbuecher:licht20:uv_17-08.png> </td>
 +
                   
 +
                    <p> numbers 1-6 are first six rows of the UV-b light approach -> look at transfections from 15.8.2013. numbers 7-12 are rows 1-6 of the red-light transfections from 15.8.2013 </p>
 +
                    <p> the system has to be improved, the controls showed the desired effect, but very weak. In the following the expression of Cas9- constructs has to be checked and improved. The tetO cri-RNA showed an insufficient binding pattern. New target sites for the plasmid have to be construct
 +
                   
 +
             
 +
              <h2> 20.8.2013 </h3>
 +
                          <h3> Construction of new cri-RNAs </h3>
 +
                                    <p> 6 new cri-RNAs are designed and ordered at sigma-aldrich. One new cri-RNA is provided by Max Stamnitz. All new cri-RNAs target pKM006! </p>
 +
                          <h3> Cloning of pIG4100; pIG4104; pIG4106; pIG4107 </h3>
 +
                          <p> Primers for new cloning- strategies have arrived. </p>
 +
                          <li> pIG4100: pKM006 is digested and the CMV-promotor from pAcGFP1 is amplified via PCR. </li>
 +
                          <li> pIG4104: pKM115 is digested with BamHI and BssHII. KRAB is amplified via PCR from pIG2001 </li>
 +
                          <li> pIG4106: pKM018 is digested with EcoRI and NotI. CIB is amplified from pSW45 </li>
 +
                          <li> pIG4107: pIG4102 is digested with EcoRI and NotI. CIB is amplified from pSW45 </li>
 +
                 
 +
              <h2> 21.8.2013 </h2>
 +
                      <h3> Cloning of pIG4100; pIG4104; pIG4106; pIG4107 </h3>
 +
                            <td> Products from digestions and PCRs are purified via gel run in 1% agarose and gelexed <td>
 +
                            <td> The digest of pKM115 for pIG4104 has not worked and is repeated executing the same protocol. </td>
 +
                            <p> Parts for pIG4100, pIG4106, pIG4107 are Gibson cloned and subsequently transformated into competent "TOP10" E.coli- cells </p>
 +
                   
 +
              <h2> 22.8.2013 </h2>
 +
                      <h3> Cloning of pIG4100; pIG4104; pIG4106; pIG4107 </h3>
 +
                            <td> Clones of Gibsons are picked and plated out. Later to be picked and test- digested. For each plasmid 2 samples were sent to GATC for sequencing. </td>
 +
                            <p> Gibson- cloning of pIG4104 is repeated with new digest and PCR-fragments. The constructs are subsequently transformated into competent "TOP10" E.coli- cells </p>
 +
                           
 +
                      <h3> Transfection control: Is Cas9-VP16 expresse? How Strong? Is it toxic? </h3>
 +
                            <p> HEK, HeLa and CHO- cells are seeded in 6-well plates </p>
 +
                           
 +
                           
 +
              <h2> 23.8.2013 </h2>
 +
                      <h3> Cloning of pIG4104 </h3>
 +
                      <td> Colonies are picked and plated out for mini and test- digest on 24.- or 25.8.13 </td>
 +
                     
 +
                  <h3> Transfection control: Is Cas9-VP16 expresse? How Strong? Is it toxic? </h3>
 +
                      <p> different DNA- concentrations are used for transfection in different cell lines. pIG1000 containing Cas9-VP16 is used. As a already tested sample, pIG8004 containing Cas9-G9a is used as positive- control. The following DNA- concentrations are transfected: 2ug; 3ug; 4ug; 6ug. One sample is tested with and addition of 0,5% DMSO into the medium attempting to boost the expression </p>
 +
                      <p> The following pattern is transfected </p>
 +
                      <td> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?media=wiki:laborbuecher:licht20:123.png> </td>
 +
 +
<h2> 24.8.2013 </h2>
 +
<h3> Oligo annealing </h3>
 +
<p> Targets T2, T3, T4, T5, T6, T7 and Max's random target were annealed.</p>
 +
<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378423065&w=500&h=264&media=wiki:laborbuecher:licht20:light_targets.png> </td>
 +
<h3> Ligation of new RNA plasmid </h3>
 +
<p> Digested pIG3010 (50,1 ng/µl) and the annealed oliges were ligated with 3 molar amount of Insert. </p>
 +
<h2> 25.8.2013 </h2>
 +
<h3> Result of ligation </h3>
 +
<p> Colonies were picked for mini preps.</p>
 +
<h2> 26.8.2013 </h2>
 +
<h3> Minipreps of targets </h3>
 +
<p> Minipreps were sent for sequencing </p>
 +
<h2> 27.8.2013 </h2>
 +
<h3> Result of sequencing </h3>
 +
<p> T2, T5 and T6 were positive. </p>
 +
<h3> Seeding of cells for UV light activation experiment </h3>
 +
<h2> 29.8.2013 </h2>
 +
<h3> Transfection of CHOs for UV light activation</h3>
 +
 +
<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378422527&w=436&h=468&media=wiki:laborbuecher:licht20:uv_light_transfection_27-08.png> </td>
 +
 +
<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378422528&w=500&h=393&media=wiki:laborbuecher:licht20:uv_transfection_calc1_27-08.png> </td>
 +
 +
<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378422528&w=500&h=355&media=wiki:laborbuecher:licht20:uv_transfection_calc2_27-08.png> </td>
 +
 +
<h2> 30.8.2013 </h2>
 +
<h3> Light start </h3>
 +
<p> UV light was started at 15.30 o clock (two plexi glass panes) </p>
 +
<h2> 31.8.2013 </h2>
 +
<h3> SEAP assay </h3>
 +
<p> SEAP assay of UV light activation was performed. </p>
 +
<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378423064&w=499&h=255&media=wiki:laborbuecher:licht20:uv_31-08.png> </td>
 +
<h2> 01.9.2013 </h2>
 +
<h3> </h3>
 +
<h2> 02.9.2013 </h2>
 +
<h3> </h3>
 +
<h2> 03.9.2013 </h2>
 +
<h3> Oligo annealing </h3>
 +
<ul>
 +
<li> 5µl Oligo forward 100 µM to get 20 µM concentration</li>
 +
<li> 5 µl Oligo reverse 100 µM to get 20 µM concentration</li>
 +
<li> 10 µl NEB buffer 2 </li>
 +
<li> 30 µl ddH2O </li>
 +
</ul>
 +
<p> Mix was heated to 98° C, Heatblock was turned off and Oligos were cooled down </p>
 +
<h3> Digest of pIG3010 </h3>
 +
<ul>
 +
<li> 10 µl pIG3010 </li>
 +
<li> 1 µl BbsI from -80° C</li>
 +
<li> 5 µl NEB buffer 2.1 </li>
 +
<li> up to 50 µl ddH2O </li>
 +
</ul>
 +
<p> Digestion was put on a gel and purified (10 ng/µl). </p>
 +
<h3> Ligation of Target 3,4 and VEGF and transformation</h3>
 +
<p> Digested pIG3010 (10 ng/µl) and the annealed oliges were ligated with 3 molar amount of Insert. </p>
 +
<h2> 04.9.2013 </h2>
 +
<h3> Result of Ligation </h3>
 +
<p> Colonies were picked </p>
 +
<h3> Gibson of pIG4104 </h3>
 +
<p> Wrong primers were used for the last pIG4104 plasmid (was seen with the last sequencing). Therefore we made a new Gibson approach. </p>
 +
<h2> 05.9.2013 </h2>
 +
<h3> Minipreps of T3, T4 and VEGF (plates) and pIG4104 (fluid culture)</h3>
 +
<p> Minipreps were send for sequencing (Primers for RNA Plasmid (in pIG3010) BGH-reverse and EBV-RP for pIG4104.) </p>
 +
<h2> 06.9.2013 </h2>
 +
<h3> Sequencing results </h3>
 +
<p> pIG4104-3 was positive, T3, T4 and VEGF RNA plasmids were negative. </p>
 +
<h3> New Ligation of T4 target into standardized RNA plasmid</h3>
 +
<p> Procedure was done by standard protocol.</p>
 +
<h3> Seeding of CHO cells</h3>
 +
<p> 4 24-well plates were ceeded with 65000 CHO cells per well.</p>
 +
<h2> 07.9.2013 </h2>
 +
<h3> Status of CHO cells </h3>
 +
<p> CHO cells were all contaminated with bacteria again. Media was plated in a 10 cm dish. Result: No contamination here.</p>
 +
<h3> seeding of HEK cells</h3>
 +
<p> HEK cells were seeded for UV & BL activation and repression experiments at around 19/19.30. 4 24-well plates and 65000 cells per well.</p>
 +
<h2> 08.09.2013 </h2>
 +
<h3> Transfection of HEKs for UV & BL activation and repression</h3>
 +
<p> HEK cells were transfected (standard protocol) at 19.30. Media was changed after 5 hours.</p>
 +
<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378679080&w=499&h=366&media=wiki:laborbuecher:licht20:bl_transfection_plan_08-09.jpg></td>
 +
<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378679080&w=500&h=371&media=wiki:laborbuecher:licht20:uv_transfection_plan_08-09.jpg></td>
 +
<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378679080&w=499&h=392&media=wiki:laborbuecher:licht20:bl_transfection_calc_08-09_1.jpg></td>
 +
<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378679080&w=499&h=356&media=wiki:laborbuecher:licht20:bl_transfection_calc_08-09_2.jpg></td>
 +
<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378679080&w=500&h=388&media=wiki:laborbuecher:licht20:uv_transfection_calc_08-09_1.jpg></td>
 +
<td/> <img class="gelpic" src=https://wiki.uni-freiburg.de/freigemintern/lib/exe/fetch.php?t=1378679080&w=499&h=354&media=wiki:laborbuecher:licht20:uv_transfection_calc_08-09_2.jpg></td> <h3> T4 RNA plasmid</h3>
 +
<p> 6 colonies were scratched out. </p>
 +
<h2> 09.09.2013 </h2>
 +
<h3> Minis of T4 </h3>
 +
<p> 3 of 6 plasmids were sent for sequencing (1-3).
 +
<h3> Light start </h3>
 +
<p> Blue light was started at 21.30 with 450 nm and unsure intesity (4 colour box, 655 intensity number). Dark control is in a light box without light on.</p>
 +
<p> UV light was started at 20.30 Dark control is in the incubator (no UV light in the lab).</p>
 +
<h2>10.09.2013</h2>
 +
<h3>Sequencing results</h3>

Revision as of 23:48, 12 September 2013

Induction - Light

29.07.2013

Plasmids that we need as templates were transformed.

30.07.2013

Primers

Primers arrived today. Primers were diluted 1:10.

Minipreps

Transformed bacteria from yesterday with our template plasmids were minipreped.

  • pKM018: 83 ng/µl
  • pKM220: 216 ng/µl
  • pKM115: 177 ng/µl
  • pKM006: 118 ng/µl
  • pSAM200: 213 ng/µl
  • pSW42: 134 ng/µl
  • pSW45: 243 ng/µl
  • pIG2004: 212 ng/µl
  • T405: 213 ng/µl

Oligo Annealing of tetO crRNA

  • Oligos tetO13 1. CC and tetO13 2. CC were annealed
  • Therefore 20 µM diluted Primes were heated on 98° C for 4 minutes and then cooled down.

PCR list, program and composition

  • aIG4102: CMV Promotor from template T405 - Primers oIG4100F+R - 589 bp
  • aIG4103: Cas9 from template pIG2004 - Primers oIG4101F+R - 4170 bp
  • aIG4104: PIF and backbone from template pKM022- Primers oIG4102F+R - 3287 bp
  • aIG4105: KRAB from template pIG2004 - Primers oIG4103F+R - 390 bp
  • aIG4106: PhyB and backbone from template pKM018 - Primers oIG4104F+R - 4922 bp
  • aIG4107: UVR8 and backbone from template pKM168 - Primers oIG4105F+R - 3921 bp
  • aIG4108: COP1 and backbone frome template pKM115 - Primers oIG4106F+R - 3972 bp
  • aIG4109: CRY2 from template pSW42 - Primers oIG4107F+R - 1591 bp
  • aIG4110: Backbone from template pKM018 - Primers oIG4108F+R - 2924 bp
  • aIG4111: Backbone with VP16 from template pKM018 - Primers oIG4109F+R - 3282 bp
  • aIG4112: CIB from template pSW45 - Primers oIG4110F+R - 593 bp
  • aIG4113: Backbone from template pKM018 - Primers oIG4111F+R - 2881 bp
  • aIG4114: CIB from template pSW45 - Primers !! oIG4110F+oIG4112R - 638 bp
µl Substance
10 Q5-HF Reaction Buffer
200 ng Template
1 Primer forward
1 Primer reverse
4 dNTPs
1 DMSO
0,5 Q5-HF Polymerase
Add to 50 H2O
Temperature [° C] Time
98 5 Min
98 30s
60 (or optimized) 30
72 30-40/kb
72 10 Min
4 hold
  • 18 cycles

PCR results

  • aIG4102: 60° C - WORKED - GelExtraction: 87,9ng/ul
  • aIG4103: 60° C - 1 of 4 WORKED, Annealing Temperature lower? - GelExtraction: 50,5ng/ul Try 56°!
  • aIG4104: 60° C - DIDN'T WORK!!! Try with 58° - Was done, has to be tested on gel.
  • aIG4105: 60° C - WORKED - GelExtraction: 70,1ng/ul I and 57,7ng/ul II
  • aIG4106: 60° C - Was done, has to be tested on gel.
  • aIG4107: 60° C - ONLY SLIGHT BAND. TRY AGAIN!
  • aIG4108: 60° C - WRONG AMPLIFICATED BAND. Try again with different temperature!
  • aIG4109: 60° C - WORKED! - GelExtraction: 176,4ng/ul
  • aIG4110: 60° C - Was done, has to be tested on gel.
  • aIG4111: 60° C - WORKED! - GelExtraction: 84,4ng/ul
  • aIG4112: 60° C - Wasn't done yet.
  • aIG4113: 60° C - Was done, has to be tested on gel.
  • aIG4114: 60° C - Wan't done yet.

2-log Marker

aIG4102 (first 4) worked, 589 bp

aIG4105 (rest) worked, 390 bp

Roth Marker

aIG4103 only one PCR worked, 4170 bp

Roth Marker

aIG4104 didn't work

aIG4111 worked, 3282 bp

2-log Marker

aIG4109 (first two bands) worked, 1591 bp

aIG4107 (next two bands) didn't work, should have been 3921 bp

aIG4108 (one band) didnt work. Wrong band was amplified, should have been 3972 bp - another band of aIG4108 can be tested on a gel, didn't have space on this one.

31.07.13

PCR results

  • aIG4103: 54°-64° C - Everything WORKED!
  • aIG4104: 58° C - Gave three bands, upper band is supposed to be right. Was confirmed on a second gel with charging the gel only with a small amount of DNA.
  • aIG4106: 60° C - Didn't work. Try temperature gradient.
  • aIG4107: 54°-64° C - Everything Worked! But not very strong bands.
  • aIG4108: 54°-64° C - Didn't Work. Try seperate PCRs.
  • aIG4110: 60° C - Didn't work. Tried temperature gradient 54°-59° C - Worked with 55° and better with every additional ° C.
  • aIG4112: 60° C - Worked!
  • aIG4113: 60° C - Worked!
  • aIG4114: 60° C - Worked!

2-log marker

UPPER SIDE

aIG4103 (2 bands) - worked!

aIG4107 (2 bands) - worked!

aIG4106 (4 bands) - didn't work with 60° C, bands are too low!

LOWER SIDE

aIG4108 temperature gradient 54°-64° C - didn't work!

2-log marker

aIG4103 temperature gradient 54°-64° C - everything worked!

2-log marker

aIG4105 (2bands) - worked!

aIG4104 (2 bands) - upper band is considered to be the right band! - Worked!

aIG4110 - didn't work with 60° C.

2-log marker

aIG4107 temperature gradient 54°-64° C - worked, but only slight bands!

2-log marker

aIG4108 (2bands) - didn't work with 60° C!

aIG4113 (2 bands) - Worked!

aIG4110 - didn't work with 60° C.

2-log marker

aIG4110 (1 band) last column of temperature gradient (59° C) - worked!

aIG4112 (2 bands) - Worked!

aIG4114 - worked!.

2-log marker

aIG4110 temperature gradient (54°-59° C) - worked from 55° C upwards!

01.08.13

PCR results

  • aIG4104: Was gelexed and size was confirmed on a gel with a smaller amount of DNA. - Worked!
  • aIG4106: Temperature gradient 54° - 59° C and excess of reverse primer oIG4104R - Worked!
  • aIG4108: Seperate PCRs were tried out. Therefore the PCR was performed with template and at a time only one primer. After these PCRs (1x with forward Primer (58° C) and 1x with reverse Primer (62° C)) both columns were mixed and another PCR was performed. - Worked, but two bands appeared, upper band is considered to be the right fragment.

2-log marker

aIG4106 temperature gradient (54°-59° C) - worked!

2-log marker

aIG4108 seperate PCRs - worked, but other amplified products can be seen, too!

Gibsons

Fragments are mixed together to a total volume of 5µl and added to 15µl Gibson Master- Mix.

pIG4101: aIG4104- 0,83µl; aIG4103- 3,96µl; H2O- 0,21µl

pIG4103: aiG4107- 2,12µl; aiG4103- 2,88µl

pIG4105: aIG4110- 1,11µl; aIG4103- 3,51µl

pIG4106: aIG4111- 0,5µl; aIG4112- 0,3µl, H2O- 4,2ul

pIG4107: aIG4113- 0,4µl; aIG4105- 0,35µl; aIG4114- 0,35µl; H2O- 3,9µl

Gibson Mixes were transformed.

Ligation of pIG4100

Digest for pIG4100

µl type
10 DNA (pKM006 or oIG4102) ~ 1000 ng
4 Cut Smart Buffer
1 EcoRI HF
1 Nhe1 HF
Add to 40µl H2O
  • Temp.: 37°C
  • Incubation time: 1,5h

2-log marker

Digested aIG4102 with 601 bp

Digested pKM006 with 6393 bp and 126 bp.

Both were gelexed: aIG4102 19 ng/µl and pKM006 40 ng/µl

Ligation and Transformation of approach pIG4100

µl type
1 T4 Ligase
2 T4 Ligase buffer
3 fold molar amount of insert
2 Backbone pKM006
1,3 Insert aIG4102
13,7 H2O
  • Temp.: RT
  • Incubation time: 15 minutes

After the ligation, 2,5 µl if this approach were transformed into E.coli and scratched out.

02.08.13

Gibson & Ligation results

Colonies could be seen on every plate except for one of the two ligation plates. Clones were picked and scratched out -> 51 Minis

Gibson

pIG4102: aIG4105- 0,4µl; aIG4106- 2,00µl; H2O- 2,6 µl

pIG4104: aiG4108- 3,2µl; aiG4105- 0,5µl; H2O- 1,3µl

Gibson Mixes were transformed.

03.08.13

Gibson results

Colonies could be seen on one of two plates, each. Colonies were scratched out for mini preps. Therefore we had clones for every construct.

Minipreps

51 Minis - concentrations between 50 and 200 ng/µl.

04.08.13

Minipreps

15 Minis.

05.08.13

Test digest

Today we made a test digest session of our 66 minis with following enzymes.

Plasmid Enzymes Size
pIG4100 EcoRI-HF & EcoRV-HF 1571 bp (Insert CMV 600bp) & 5417 bp
pIG4101 EcoRV-HF & NotI-HF 1952 bp (half the Cas9) & 5456 bp
pIG4102 EcoRI-HF & NotI-HF 1966 bp (with PhyB) & 3274 bp
pIG4103 BamHI-HF & XhoI 5337 bp (Cas9, UVR8) & 2682 bp
pIG4104 NotI-HF & BamHI-HF 1451 bp (with COP1) & 2839 bp
pIG4105 EcoRI-HF & EcoRV-HF 3040 bp (Half the Cas9 and half CRY2) & 5529 bp
pIG4106 NotI-HF & XbaI 970 bp (CIB, VP16) & 2833 bp
pIG4107 PstI-HF & NotI-HF 976 bp (CIB, KRAB) & 2821 bp
µl type
~ 100-200 ng DNA
1 Cut Smart buffer
0,5 Enzyme 1
0,5 Enzyme 2
Add to 10µl H2O
  • Temp.: 37°C
  • Incubation time: 2h

2-log marker

Upper bands: pIG4100, expected sizes: Plasmid 3 and 5 were send to GATC

Bottom bands: pIG4101, expected sizes: Plasmid 4 and 7 were send to GATC

2-log marker

pIG4102, expected sizes, Plasmid 1 and 9 were send to GATC.

2-log marker

Upper bands left side: pIG4104, expected sizes: Plasmid 1 and 4 were send to GATC

Upper bands right side: pIG4105, expected sizes: Plasmid 1 and 6 were send to GATC

2-log marker

Upper bands: pIG4106,
not expected sizes: Plasmid 3 was send to GATC

Bottom bands: pIG4107, expected sizes: Plasmid 1 and 4 were send to GATC

For the sequencing we used GATC Primers: CMV-F for pIG4100 and EBV-RP (binds in pSV40) for all other plasmids.

Insertion of crRNA into RNA Plasmid

  1. First RNA Plasmid backbone was digested with BbsI for 3 hours. Enzyme was taken out of - 80° C.
  2. Then it was put on a gel and gelexed. Concentration: 50,5ng/ug. Backbone from Hormone- Group: 8,7ng/ul
  3. Ligation: Digested backbone from Hormone- Induction and self- digested backbone was used. Molar ratio of backbone : insert - 1 : 3.
  4. Mix: Backbone; Insert, 1ul T4- ligase, 2ul T4-ligase-buffer, H2O up to a total volume of 20ul
  5. Incubated for 15min at RT
  6. Transformed and scratched out.

06.08

Sequencing results

Sequencing results were
positive for pIG4101, pIG4102, pIG4103, pIG4105, pIG4106 and pIG4107
(sequenced with EBV-RP -> SV40 Terminator). pIG4100 didn't have the CMV promotor as an insert (-> new ligation) and pIG4104 didn't contain KRAB, although KRAB worked with pIG4102 (new Primers?)

Re-Trafos and inoculation for midi-preps

The positive Plasmids and already existing plasmids pKM018 and pKM115 were retransformed and put into 150 ml LB with 150 µl Ampicillin and incubated at 37° C over night.

New Primers for pIG4104

New Primers were designed for pIG4104.

Ligation of pIG4100

Ligation of pIG4100 was done again like on 31.07.13, only the ratio of backbone and insert was changed (3 µl backbone and 1,5 µl Insert).

2-log marker

Left band: Backbone, Right band: Inser

Result of Ligation of RNA Plasmid

There were clones on every plate. They were scratched out for minipreps.

New sequencing

Plasmids were send to GATC with different primers. Cas9 was sequenced completely in pIG4101.7. Other pIG4104 were screened for insertion of KRAB.

7.8.2013

Midipreps of pKM017; pIG4101.7 (?), pIG4102.1, pIG4103, pKM115, pIG4105.5, pIG4106.3, pIG4107.4

All midipreps were successfull

Concentrations:

pKM018: 474 ng/ul

pIG4101: 485 ng/ul

pIG4102: 528 ng /ul

pIG4103: 521 ng/ul

pKM115: 517 ng/ul

pIG4105.1: 492 ng /ul

pIG4106: 567 ng/ul

pIG4107: 476 ng /ul

Minipreps of RNA Plasmid pIG4201 and pIG4202

Minis were send to GATC. 3 of ligation with digested Backbone from Michi and tetO1 and 3 with tetO2. And 3 of ligation with newly digested Backbone from Gabi and tetO1 and 3 with tetO2.

Result of Ligation of pIG4100

There were clones on every plate. They were scratched out for minis.

8.8.2013

Sequencing of RNA Plasmid

1 of the 6 pIG4201 and 4 of the 6 pIG4202 were positiv with inserted crRNA.

Sequencing Order

Sequencing of pIG4107_2 and pIG4107_3 with Primer EBV-RP

Minis of pIG4100

12 Minis were prepped.

Re-Trafo for inoculation for Midi Preps

The following plasmids were retransformed: pIG1000, pIG4201, pIG4202

9.8.2013

Midis of ...

Test digest of pIG4100

10.8.2013

Seeding of CHO cells

CHO- cells were seeded: 70.000 cells per well on 24 well- plates in HTS-medium

11.8.2013

Transfection of CHO cells

Protocol for 24- well plates:

1. Mix 40 µl Opti-MEM + 1.5 µl PEI-solution in a 1.5 ml Eppi

2.Prepare in another Eppi 0.5 µg of the DNA of interest

3.Add DNA to former Eppi, vortex thoroughly and incubate 15 min at RT

4.Resuspend solution gently and spread them drop-wise to the cells in the dish

5.Swing the dish in axis-form

6.Change of media 5h post transfection

12.8.2013

PCB- treatment of CHO-cells and illumination

Cells are treated with HTS-medium containing 15uM of phycocyanobilin

illumination started at 16.15. with 20uE light- quantity

plate 1 with activating red light (660nm)

plate 2 with inctivating far-red light (740nm), control

construction of pIG4104

pKM115 was digested with BamHI and BssHII for 4h. KRAB was amplified from pIG2004 with oIG4103FII and oIG4103RII

Subsequently the fragments were assembled via Gibson-cloning and transformed into E.coli

12.8.2013

Gibson results

Cell-colonies are picked and plated out anew

A mistake in the cloning strategy was discovered, new primers are designed

13.8.2013

Gibson results

CHO- cells are illuminated

14.8.2013

Seeding of CHO-cells

CHO- cells were seeded: 70.000 cells per well on 24 well- plates in HTS-medium

SEAP-measurements of illuminated cells

The results are all negative except for the positive control (-> see transfection Nr.6 of 11.8.2013)

The interaction with the cri-RNA may be responsible. The experiment is repeated with new target- sites

15.8.2013

Transfection of CHO-cells for red light and UV tests

The wiki- transfection protocoll is used:

1. Mix 40 µl Opti-MEM + 1.5 µl PEI-solution in a 1.5 ml Eppi

2.Prepare in another Eppi 0.5 µg of the DNA of interest

3.Add DNA to former Eppi, vortex thoroughly and incubate 15 min at RT

4.Resuspend solution gently and spread them drop-wise to the cells in the dish

5.Swing the dish in axis-form

6.Change of media 5h post transfection

The following pattern is transfected in 24- well plates:

16.8.2013

UV-b and red-/ far red light illumination of transfected cells from 15.8.2013

Cells for red- light illumination are illuminated with 20uE; 660nm or 720nm for 24h. Illumination is started at 14.20

Cells for UV-b light illumination are illiminated with 5uE; 311nm for 24h

17.8.2013

SEAP-measurements taken from illuminated- and untreated cells.

the following protocoll was obeyed for the whole process

  • 200 µl of cell supernatant were transfered form the 24-well plate into one 96-well plate (round bottom)
  • Incubation for 30 min at 65 °C.
  • Centrifugation for 1 min at 1250 g.
  • 100 µl of SEAP buffer were filled in the wells of a 96 well plate (flat bottom).
  • Addition of 80 µl supernatant from the round bottom plate to each well with SEAP buffer.
  • Addition of 20 µl pNPP (substrate).
  • 96 plate was immediately placed in the blade reader.

    • Spectroscopic measurement every minute for 120 times (2h); wavelenght 405 nm.

    numbers 1-6 are first six rows of the UV-b light approach -> look at transfections from 15.8.2013. numbers 7-12 are rows 1-6 of the red-light transfections from 15.8.2013

    the system has to be improved, the controls showed the desired effect, but very weak. In the following the expression of Cas9- constructs has to be checked and improved. The tetO cri-RNA showed an insufficient binding pattern. New target sites for the plasmid have to be construct

    20.8.2013

    Construction of new cri-RNAs

    6 new cri-RNAs are designed and ordered at sigma-aldrich. One new cri-RNA is provided by Max Stamnitz. All new cri-RNAs target pKM006!

    Cloning of pIG4100; pIG4104; pIG4106; pIG4107

    Primers for new cloning- strategies have arrived.

  • pIG4100: pKM006 is digested and the CMV-promotor from pAcGFP1 is amplified via PCR.
  • pIG4104: pKM115 is digested with BamHI and BssHII. KRAB is amplified via PCR from pIG2001
  • pIG4106: pKM018 is digested with EcoRI and NotI. CIB is amplified from pSW45
  • pIG4107: pIG4102 is digested with EcoRI and NotI. CIB is amplified from pSW45

    • 21.8.2013

    Cloning of pIG4100; pIG4104; pIG4106; pIG4107

    Products from digestions and PCRs are purified via gel run in 1% agarose and gelexed The digest of pKM115 for pIG4104 has not worked and is repeated executing the same protocol.

    Parts for pIG4100, pIG4106, pIG4107 are Gibson cloned and subsequently transformated into competent "TOP10" E.coli- cells

    22.8.2013

    Cloning of pIG4100; pIG4104; pIG4106; pIG4107

    Clones of Gibsons are picked and plated out. Later to be picked and test- digested. For each plasmid 2 samples were sent to GATC for sequencing.

    Gibson- cloning of pIG4104 is repeated with new digest and PCR-fragments. The constructs are subsequently transformated into competent "TOP10" E.coli- cells

    Transfection control: Is Cas9-VP16 expresse? How Strong? Is it toxic?

    HEK, HeLa and CHO- cells are seeded in 6-well plates

    23.8.2013

    Cloning of pIG4104

    Colonies are picked and plated out for mini and test- digest on 24.- or 25.8.13

    Transfection control: Is Cas9-VP16 expresse? How Strong? Is it toxic?

    different DNA- concentrations are used for transfection in different cell lines. pIG1000 containing Cas9-VP16 is used. As a already tested sample, pIG8004 containing Cas9-G9a is used as positive- control. The following DNA- concentrations are transfected: 2ug; 3ug; 4ug; 6ug. One sample is tested with and addition of 0,5% DMSO into the medium attempting to boost the expression

    The following pattern is transfected

    24.8.2013

    Oligo annealing

    Targets T2, T3, T4, T5, T6, T7 and Max's random target were annealed.

    Ligation of new RNA plasmid

    Digested pIG3010 (50,1 ng/µl) and the annealed oliges were ligated with 3 molar amount of Insert.

    25.8.2013

    Result of ligation

    Colonies were picked for mini preps.

    26.8.2013

    Minipreps of targets

    Minipreps were sent for sequencing

    27.8.2013

    Result of sequencing

    T2, T5 and T6 were positive.

    Seeding of cells for UV light activation experiment

    29.8.2013

    Transfection of CHOs for UV light activation

    30.8.2013

    Light start

    UV light was started at 15.30 o clock (two plexi glass panes)

    31.8.2013

    SEAP assay

    SEAP assay of UV light activation was performed.

    01.9.2013

    02.9.2013

    03.9.2013

    Oligo annealing

    • 5µl Oligo forward 100 µM to get 20 µM concentration
    • 5 µl Oligo reverse 100 µM to get 20 µM concentration
    • 10 µl NEB buffer 2
    • 30 µl ddH2O

    Mix was heated to 98° C, Heatblock was turned off and Oligos were cooled down

    Digest of pIG3010

    • 10 µl pIG3010
    • 1 µl BbsI from -80° C
    • 5 µl NEB buffer 2.1
    • up to 50 µl ddH2O

    Digestion was put on a gel and purified (10 ng/µl).

    Ligation of Target 3,4 and VEGF and transformation

    Digested pIG3010 (10 ng/µl) and the annealed oliges were ligated with 3 molar amount of Insert.

    04.9.2013

    Result of Ligation

    Colonies were picked

    Gibson of pIG4104

    Wrong primers were used for the last pIG4104 plasmid (was seen with the last sequencing). Therefore we made a new Gibson approach.

    05.9.2013

    Minipreps of T3, T4 and VEGF (plates) and pIG4104 (fluid culture)

    Minipreps were send for sequencing (Primers for RNA Plasmid (in pIG3010) BGH-reverse and EBV-RP for pIG4104.)

    06.9.2013

    Sequencing results

    pIG4104-3 was positive, T3, T4 and VEGF RNA plasmids were negative.

    New Ligation of T4 target into standardized RNA plasmid

    Procedure was done by standard protocol.

    Seeding of CHO cells

    4 24-well plates were ceeded with 65000 CHO cells per well.

    07.9.2013

    Status of CHO cells

    CHO cells were all contaminated with bacteria again. Media was plated in a 10 cm dish. Result: No contamination here.

    seeding of HEK cells

    HEK cells were seeded for UV & BL activation and repression experiments at around 19/19.30. 4 24-well plates and 65000 cells per well.

    08.09.2013

    Transfection of HEKs for UV & BL activation and repression

    HEK cells were transfected (standard protocol) at 19.30. Media was changed after 5 hours.

    T4 RNA plasmid

    6 colonies were scratched out.

    09.09.2013

    Minis of T4

    3 of 6 plasmids were sent for sequencing (1-3).

    Light start

    Blue light was started at 21.30 with 450 nm and unsure intesity (4 colour box, 655 intensity number). Dark control is in a light box without light on.

    UV light was started at 20.30 Dark control is in the incubator (no UV light in the lab).

    10.09.2013

    Sequencing results

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