Team:Toronto/Notebook

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{{CSS/Main}}
{{CSS/Main}}
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{{Templates/head}}
<html>
<html>
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<div id="bg"></div>
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<div id="splash-wrapper">
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<section id="splash" style="position:absolute;">
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<div class="container">
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<!--<h1><span>Biocontainment: a novel approach</span></h1>-->
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</div>
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</section>
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<nav>
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<div class="container" style="position:relative;">
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<ul id="navbar">
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                                <div id="tabs" style="display:inline-block; width:80%; position:absolute; bottom:0;">
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<li id="home">
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<a href='https://2014.igem.org/Team:Toronto#navbar' scroll-on-click><i class="fa fa-home"></i>Home</a>
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</li>
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<li id="team">
 +
<a href='https://2014.igem.org/Team:Toronto/Team#navbar' scroll-on-click><i class="fa fa-users"></i> Team</a>
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</li>
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<li id="project">
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<a href='https://2014.igem.org/Team:Toronto/Project#navbar' scroll-on-click><i class="fa fa-gears"></i> Project</a>
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</li>
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<li id="parts">
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<a href='https://2014.igem.org/Team:Toronto/Parts#navbar' scroll-on-click><i class="fa fa-wrench"></i> Parts</a>
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</li>
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<li id="modeling">
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<a href='https://2014.igem.org/Team:Toronto/Modeling#navbar' scroll-on-click>
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<i class="fa fa-bar-chart"></i> Modeling</a>
 +
</li>
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<li class="active" id="notebook">
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<a href='https://2014.igem.org/Team:Toronto/Notebook#navbar' scroll-on-click><i class="fa fa-book"></i> Notebook</a>
 +
</li>
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                                <li id="humanpractices">
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<a href='https://2014.igem.org/Team:Toronto/HumanPractices#navbar' scroll-on-click><i class="fa fa-child"></i> Human Practices</a>
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</li>
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<li id="safety">
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<a href='https://2014.igem.org/Team:Toronto/Safety#navbar' scroll-on-click><i class="fa fa-exclamation-triangle"></i> Safety</a>
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</li>
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<li id="attributions">
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<a href='https://2014.igem.org/Team:Toronto/Attributions#navbar' scroll-on-click><i class="fa fa-asterisk"></i> Attributions</a>
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</li>
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                                <div style="clear:both;"></div>
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                                </div>
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        <div id="igemlogo" style="display:inline-block; float:right;">
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                            <a href="2014.igem.org"><img style="height:100px;" src="https://static.igem.org/mediawiki/2014/8/84/Igemlogo_300px2.png"> </a>
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                        </div>
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                            </ul>
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</div>
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</nav>
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</div>
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<!--main content -->
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<section id="main">
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<table width="70%" align="center">
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<div id="main-inner">
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<div id="view" ng-view data-autoscroll="true">
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<div class="first container-wrapper">
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<!--welcome box -->
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<div class="container">
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<tr>
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<ul>
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<td style="border:1px solid black;" colspan="3" align="center" height="150px" bgColor=#FF404B>
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<li class="week">
-
<h1 >WELCOME TO iGEM 2014! </h1>
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<h2><span>Week 1</span></h2>
-
<p>Your team has been approved and you are ready to start the iGEM season!
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<ul class="thingsdone">
-
<br>On this page you can document your project, introduce your team members, document your progress <br> and share your iGEM experience with the rest of the world! </p>
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<li>
-
<br>
+
picked single MG1655 and pTRC99A strains into liquid culture,
-
<p style="color:#E7E7E7"> <a href="https://2014.igem.org/wiki/index.php?title=Team:Toronto/Notebook&action=edit"style="color:#FFFFFF"> Click here  to edit this page!</a> </p>
+
incubated overnight (pTRC99A in LB+Amp)
-
</td>
+
</li>
-
</tr>
+
<ul>
-
 
+
<li>
-
<tr> <td colspan="3"  height="5px"> </td></tr>
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miniprepped pTRC, O/N culture of MG1655
-
<!-- end welcome box -->
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</li>
-
<tr>  
+
<li>
-
 
+
MG1655 did not grow on antibiotic plates, so stocks are good!
-
<!--navigation menu -->
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</li>
-
<td align="center" colspan="3">
+
</ul>
-
 
+
<li>
-
<table  width="100%">
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prepped DNA for transformation – pCas9 and RFP reporter
-
<tr heigth="15px"></tr>
+
</li>
-
<tr heigth="75px">  
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<li>
-
 
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made MG1655 electrocompetent –
-
 
+
electroporate and plated transformants, incubated overnight
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<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>
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</li>
-
<a href="https://2014.igem.org/Team:Toronto"style="color:#000000">Home </a> </td>
+
</ul>
-
 
+
</li>
-
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>  
+
<li class="week">
-
<a href="https://2014.igem.org/Team:Toronto/Team"style="color:#000000"> Team </a> </td>
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<h2><span>Week 2</span></h2>
-
 
+
<ul class="thingsdone">
-
<td style="border:1px solid black;" align="center"  height ="45px"  onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>  
+
<li>
-
<a href="https://igem.org/Team.cgi?year=2014&team_name=Toronto"style="color:#000000"> Official Team Profile </a></td>
+
transformation of RFP, pCas9, GFP, fmt into NEB5alpha
-
 
+
</li>
-
<td style="border:1px solid black" align="center"  height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>
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<ul>
-
<a href="https://2014.igem.org/Team:Toronto/Project"style="color:#000000"> Project</a></td>
+
<li>
-
 
+
transformants plated and incubated overnight
-
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>  
+
</li>
-
<a href="https://2014.igem.org/Team:Toronto/Parts"style="color:#000000"> Parts</a></td>
+
<li>
-
 
+
then inoculated into liquid culture, incubated overnight
-
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>  
+
</li>
-
<a href="https://2014.igem.org/Team:Toronto/Modeling"style="color:#000000"> Modeling</a></td>
+
</ul>
-
 
+
<li>
-
<td style="border:1px solid black;" align="center" height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>
+
overnight culture of MG1655 and pTRC99A
-
<a href="https://2014.igem.org/Team:Toronto/Notebook"style="color:#000000"> Notebook</a></td>
+
</li>
-
 
+
<li>
-
<td style="border:1px solid black;" align="center"  height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>  
+
Miniprepped RFP and pTRC99A
-
<a href="https://2014.igem.org/Team:Toronto/Safety"style=" color:#000000"> Safety </a></td>
+
</li>
-
 
+
<ul>
-
<td style="border:1px solid black;" align="center"  height ="45px" onMouseOver="this.bgColor='#d3d3d3'" onMouseOut="this.bgColor='#e7e7e7'" bgColor=#e7e7e7>  
+
<li>tested restriction enzymes EcoRI, Xbal, SpeI, Pstl
-
<a href="https://2014.igem.org/Team:Toronto/Attributions"style="color:#000000"> Attributions </a></td>
+
on miniprep and ran on gel w/ uncut as control
-
 
+
</li>
-
 
+
<li>
-
<td align ="center"> <a href="https://2014.igem.org/Main_Page"> <img src="https://static.igem.org/mediawiki/igem.org/6/60/Igemlogo_300px.png" width="55px"></a> </td>
+
cut plasmids were too faint to see – need more
-
</tr>
+
concentrated DNA in preps!
-
</table>
+
</li>
-
 
+
</ul>
-
 
+
<li>
-
</tr>
+
O/N cultures of RFP, pCas9, GFP from transformation
-
 
+
</li>
-
 
+
<li>
-
</tr>
+
miniprep of O/N cultures of transformants and pTRC99A
-
+
</li>
-
 
+
</ul>
-
 
+
<li class="week">
-
 
+
<h2><span>Week 3</span></h2>
-
 
+
<ul class="thingsdone">
-
</td>
+
<li>
-
 
+
miniprep again, greater volume (3mL)
-
<tr> <td colspan="3"  height="15px"> </td></tr>
+
for RFP, GFP, Cas9, pTRC99A + nanodrop
-
<tr><td bgColor="#e7e7e7" colspan="3" height="1px"> </tr>
+
</li>
-
<tr> <td colspan="3" height="5px"> </td></tr>
+
<ul>
-
 
+
<li>
-
 
+
digest product (w SpeI, EcoRI, cutsmart) and run on gel
-
<!--requirements section -->
+
</li>
-
<tr><td colspan="3"> <h3>Notebook</h3></td></tr>
+
</ul>
-
</tr>
+
<li>
-
 
+
O/N culture from plate of extra biobrick, RFP, MG1655
-
 
+
</li>
-
<tr>
+
<li>
-
<td width="45%" valign="top">  
+
start RFP and MG1655 liquid cultures for the RFP
-
<p>You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well. </p>
+
spectrum measurements in the afternoon
-
</td>
+
</li>
-
 
+
<ul>
-
<td></td>
+
<li>
-
<td></td>
+
RFP grew slowly and was transformed into NEB5alpha instead of MG1655
-
</tr>
+
</li>
-
 
+
</ul>
 +
<li>
 +
digest 8 samples from yesterday’s miniprep of 8 biobrick cultures,
 +
load and run gel of digest
 +
</li>
 +
<li>
 +
oligos and gblocks arrived
 +
</li>
 +
<li>
 +
did PCR of pCas9 and pTRC99A chunks
 +
</li>
 +
<li>
 +
Started MG1655 liquid culture for making chemically competent
 +
</li>
 +
<ul>
 +
<li>
 +
make chemically competent cells, transform RFP into them,
 +
PCR purification, run on gel
 +
</li>
 +
</ul>
 +
</ul>
 +
<li class="week">
 +
<h2><span>Week 4</span></h2>
 +
<ul class="thingsdone">
 +
<li>
 +
miniprepped pCas9 + pTRC99A (w 7mL of cells)
 +
</li>
 +
<li>
 +
did gel extraction – cut out pCas9 + pTRC digests –
 +
PCR both as well as nanodrop
 +
</li>
 +
<li>
 +
Mon/Tues = PCR w mastermix,
 +
Wed = make comp MG1655 and heatshock RFP,
 +
Thurs/Fri = confirm RFP and Marafini protocol/golden-gate assembly
 +
</li>
 +
<ul>
 +
<li>
 +
PCR from the Friday before did not work
 +
</li>
 +
<li>
 +
so do lots of minipreps from 10mL cultures and start over, use larger volumes for digests
 +
</li>
 +
DNA eluted into collection tube but saw salt contamination when nanodropped therefore redo miniprep…
 +
</li>
 +
<li>
 +
miniprepped pTRC and pCas9 and then nanodropped –
 +
results were good so created gel for next day, as well as a digestion
 +
</li>
 +
<li>
 +
digested/ran the gel/extracted, will nanodrop gel extraction – if worked, try PCR again
 +
</li>
 +
</ul>
 +
</ul>
 +
<li class="week">
 +
<h2><span>Week 5</span></h2>
 +
<ul class="thingsdone">
 +
<li>
 +
Wed. minipreps were good (however possibly from just high free nucleotides), gel extraction failed (no DNA at the end and a lot of salt contamination) – so just PCR from miniprepped plasmids
 +
</li>
 +
<li>
 +
digest and run gel of digest at the same time!
 +
</li>
 +
<ul>
 +
<li>
 +
PCR of pTRC and pCas9 – have also been digested
 +
</li>
 +
<li>
 +
ran gel! 5 part sample, 1 part loading dye
 +
</li>
 +
</ul>
 +
<li>
 +
if PCR worked, digest PCR product w DpnI – got nothing for pCas9 though on the gel (to be re-PCR-ed), but bright bands for pTRC
 +
</li>
 +
<li>
 +
competent MG1655 also done
 +
</li>
 +
<li>
 +
PCR of miniprep C1 #1 of cas9… did 10 x 10uL reaction using gradient temps in 58-68 range
 +
</li>
 +
<ul>
 +
<li>
 +
run on gel to see if PCR worked
 +
</li>
 +
<li>
 +
PCR gel showed 2 weak bands – either not enough DNA or PCR failed
 +
</li>
 +
</ul>
 +
<li>
 +
heat shock transformation for MG1655 w RFP
 +
plasmid but very few colonies on CM plate
 +
</li>
 +
</ul>
 +
<li class="week">
 +
<h2><span>Week 6</span></h2>
 +
<ul class="thingsdone">
 +
<li>
 +
PCR product (???) run on gel, pick most successful PCR
 +
</li>
 +
<li>
 +
DpnI digest following protocol in NEB Gibson manual
 +
Transform RFP & MG1655, O/N cultures
 +
</li>
 +
<li>
 +
pCas9PCR product visible on gel
 +
</li>
 +
<li>
 +
DpnI digest + PCR cleanup of digest (ended up not having enough DNA)
 +
</li>
 +
<li>
 +
plating of transformed RFP into DH5a
 +
</li>
 +
<li>
 +
processed PCR – ran on gel, DpnI digest also ran on gel, PCR cleanup, nanodrop (worked well)
 +
</li>
 +
<li>
 +
needed to transform pCas9+spacer plasmid & RFP DH5a
 +
</li>
 +
</ul>
 +
<li class="week">
 +
<h2><span>Week 7/8</span></h2>
 +
<ul class="thingsdone">
 +
<li>
 +
transform RFP & BL21(DE3)
 +
</li>
 +
<li>
 +
PCR of pCas9, pTRC99A
 +
</li>
 +
<li>
 +
Gibson – pick 8 colonies, make O/N colonies, try to PCR form reaction to get 4-fragment superchunk
 +
</li>
 +
<li>
 +
Maraffini – use Anderson high for 4 fragment
 +
(phosphorylation + annealing of spacers if we get a lot of DNA), otherwise using Aug 18 miniprep do a BsaI digest
 +
</li>
 +
<ul>
 +
<li>
 +
got colonies from Gibson
 +
</li>
 +
</ul>
 +
<li>
 +
3 things in parallel:
 +
</li>
 +
<ul>
 +
<li>
 +
miniprepped plasmids from Gibson transformants from O/N liquid cultures (need to be sent for sequencing)
 +
, plasmids digested w E+P and to be run on gel
 +
</li>
 +
<li>
 +
making competent MG1655 – transform RFP into it
 +
</li>
 +
<li>
 +
tried BsaI digestion and gel extraction on Stanford-brown pCas9 (for Marafini/Golden-gate),
 +
does not seem to work even w large volumes
 +
</li>
 +
</ul>
 +
<li>
 +
look at plates from transformation, run E+P digests on gel – if the digests look wrong size, do colony PCR on rest of Gibson colonies on plates
 +
</li>
 +
<ul>
 +
<li>
 +
use fwd/rev primers for cas9 and p!TRC chunks
 +
</li>
 +
<li>
 +
if correct, also PCR the 3-fragment superchunk from the miniprep plasmid by using Cas9 fwd with p!TRC rev primers
 +
</li>
 +
</ul>
 +
<li>
 +
try a gel-extraction protocol…
 +
</li>
 +
</ul>
 +
<li class="week">
 +
<h2><span>Week 9</span></h2>
 +
<ul class="thingsdone">
 +
<li>
 +
Main thing – <b>GIBSON ASSEMBLY</b>
 +
</li>
 +
<li>
 +
screened 8 colonies – miniprepped, EcoRI + PstI digest & wrong product, non-specific assembly
 +
</li>
 +
<ul>
 +
<li>
 +
extend gblocks (Anderson high + tracr) so more overlap, try Gibson again
 +
</li>
 +
</ul>
 +
<li>
 +
did digests of plasmids w one restriction enzyme instead of double digest + run gel (for less confusing results) – used miniprep #1-4
 +
</li>
 +
<li>
 +
made O/N cultures from first Gibson reaction #9-15 – miniprepped and streak out plates
 +
</li>
 +
<li>
 +
minipreps #5-8 from earlier digested w EcoRI and PstO (separately), run on gel
 +
</li>
 +
<li>
 +
take 2 minipreps, do 3 reactions on each – EcoRI only, PstI only, both EcoRI + PstI
 +
</li>
 +
</ul>
 +
<li class="week">
 +
<h2><span>Week 10</span></h2>
 +
<ul class="thingsdone">
 +
<li>
 +
miniprep out RFP plasmid to be used for putting biobricks into pSB1C3, will want to screen for non-red colonies at the end of the process
 +
</li>
 +
<li>
 +
unable to do digest because nanoprep of all 3 minipreps was too low
 +
</li>
 +
<li>
 +
primers arrived – ran PCR w new primers
 +
</li>
 +
<ul>
 +
<li>
 +
also digested RPF
 +
</li>
 +
<li>
 +
run digests on gel (1/2 thickness)
 +
</li>
 +
</ul>
 +
<li>
 +
ran the original gblocks w extended gblocks –
 +
not enough of original gblocks to show up on gel
 +
</li>
 +
<li>
 +
combined PCR reactions of tracr gblocks into one,
 +
same with Anderson High PCR products
 +
</li>
 +
<ul>
 +
<li>
 +
purified both
 +
</li>
 +
<li>
 +
PCR purified Cas9 array from a PCR in August
 +
</li>
 +
<li>
 +
purified p!TRC chunk found
 +
</li>
 +
<li>
 +
all 4 to be used for Gibson assembly!
 +
</li>
 +
</ul>
 +
<li>
 +
glycerol stocked 3 tubes of colonies from first
 +
Gibson attempt (#3, 4, 7)
 +
</li>
 +
<li>
 +
transformed Gibson in NEB5alpha, ½ of each reaction
 +
tube into a total of 4 NEB5alpha – 4 tubes plated
 +
onto 2 plates each = total 8 plates incubated
 +
</li>
 +
<li>
 +
on Friday, found colonies from the Gibson assembly and
 +
did colony PCR
 +
</li>
 +
<li>
 +
colony PCR primers were designed to cover the seams
 +
where the 4 chunks joined
 +
</li>
 +
<li>
 +
mixed the template DNA and biobrick primers for the
 +
Cas9-array chunk, but did not add the master mix yet
 +
(done later)
 +
</li>
 +
</ul>
 +
<li class="week">
 +
<h2><span>Week 11</span></h2>
 +
<ul class="thingsdone">
 +
<li>
 +
ran Gibson colony PCR on gel – colonies #3 + 6 have correct bands, started O/N cultures of them w cell suspensions from colony PCR
 +
</li>
 +
<li>
 +
miniprep and glycerol stock them (low copy plasmid – do 3uL miniprep)
 +
</li>
 +
<li>
 +
PCR of cas9 chunk – run on gel, see if band is at 4595
 +
</li>
 +
<li>
 +
using RFP miniprep from earlier, digest w EcoRI and PstI
 +
</li>
 +
<ul>
 +
<li>
 +
linearised plasmid digested same way (if DNA dissolved – was left sitting overnight to dissolve)
 +
</li>
 +
</ul>
 +
<li>
 +
nothing on gel at correct size…. only used 1uL of DNA for gel though
 +
</li>
 +
<li>
 +
digest ~1000ng of Gibson miniprep from 2 colonies w EcoRI and PstI, run on gel
 +
</li>
 +
<li>
 +
run 5uL of tube labelled C6*
 +
</li>
 +
<li>
 +
cas9-array chunk biobrick extension PCR barely worked – not enough DNA after PCR purification
 +
but tracr chunk biobrick PCR worked
 +
</li>
 +
<ul>
 +
<li>
 +
digested/ligated into pSB1C3, transformed into competent cells
 +
</li>
 +
<li>
 +
Gibson colonly #3 didn’t assemble correctly, only produced 1 band from E+P digestion
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transformed ligation into NEB5alpha – after 2 hour (actual = 10 min) incubation in SOB, pipetted into 2mL LB Cm and grew overnight
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</li>
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miniprepped to see if it grew and packed it up for shipping!
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2014 University of Toronto iGEM
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Latest revision as of 01:47, 18 October 2014


  • Week 1

    • picked single MG1655 and pTRC99A strains into liquid culture, incubated overnight (pTRC99A in LB+Amp)
      • miniprepped pTRC, O/N culture of MG1655
      • MG1655 did not grow on antibiotic plates, so stocks are good!
    • prepped DNA for transformation – pCas9 and RFP reporter
    • made MG1655 electrocompetent – electroporate and plated transformants, incubated overnight

  • Week 2

    • transformation of RFP, pCas9, GFP, fmt into NEB5alpha
      • transformants plated and incubated overnight
      • then inoculated into liquid culture, incubated overnight
    • overnight culture of MG1655 and pTRC99A
    • Miniprepped RFP and pTRC99A
      • tested restriction enzymes EcoRI, Xbal, SpeI, Pstl on miniprep and ran on gel w/ uncut as control
      • cut plasmids were too faint to see – need more concentrated DNA in preps!
    • O/N cultures of RFP, pCas9, GFP from transformation
    • miniprep of O/N cultures of transformants and pTRC99A

  • Week 3

    • miniprep again, greater volume (3mL) for RFP, GFP, Cas9, pTRC99A + nanodrop
      • digest product (w SpeI, EcoRI, cutsmart) and run on gel
    • O/N culture from plate of extra biobrick, RFP, MG1655
    • start RFP and MG1655 liquid cultures for the RFP spectrum measurements in the afternoon
      • RFP grew slowly and was transformed into NEB5alpha instead of MG1655
    • digest 8 samples from yesterday’s miniprep of 8 biobrick cultures, load and run gel of digest
    • oligos and gblocks arrived
    • did PCR of pCas9 and pTRC99A chunks
    • Started MG1655 liquid culture for making chemically competent
      • make chemically competent cells, transform RFP into them, PCR purification, run on gel

  • Week 4

    • miniprepped pCas9 + pTRC99A (w 7mL of cells)
    • did gel extraction – cut out pCas9 + pTRC digests – PCR both as well as nanodrop
    • Mon/Tues = PCR w mastermix, Wed = make comp MG1655 and heatshock RFP, Thurs/Fri = confirm RFP and Marafini protocol/golden-gate assembly
      • PCR from the Friday before did not work
      • so do lots of minipreps from 10mL cultures and start over, use larger volumes for digests
        • DNA eluted into collection tube but saw salt contamination when nanodropped therefore redo miniprep…
      • miniprepped pTRC and pCas9 and then nanodropped – results were good so created gel for next day, as well as a digestion
      • digested/ran the gel/extracted, will nanodrop gel extraction – if worked, try PCR again

  • Week 5

    • Wed. minipreps were good (however possibly from just high free nucleotides), gel extraction failed (no DNA at the end and a lot of salt contamination) – so just PCR from miniprepped plasmids
    • digest and run gel of digest at the same time!
      • PCR of pTRC and pCas9 – have also been digested
      • ran gel! 5 part sample, 1 part loading dye
    • if PCR worked, digest PCR product w DpnI – got nothing for pCas9 though on the gel (to be re-PCR-ed), but bright bands for pTRC
    • competent MG1655 also done
    • PCR of miniprep C1 #1 of cas9… did 10 x 10uL reaction using gradient temps in 58-68 range
      • run on gel to see if PCR worked
      • PCR gel showed 2 weak bands – either not enough DNA or PCR failed
    • heat shock transformation for MG1655 w RFP plasmid but very few colonies on CM plate

  • Week 6

    • PCR product (???) run on gel, pick most successful PCR
    • DpnI digest following protocol in NEB Gibson manual Transform RFP & MG1655, O/N cultures
    • pCas9PCR product visible on gel
    • DpnI digest + PCR cleanup of digest (ended up not having enough DNA)
    • plating of transformed RFP into DH5a
    • processed PCR – ran on gel, DpnI digest also ran on gel, PCR cleanup, nanodrop (worked well)
    • needed to transform pCas9+spacer plasmid & RFP DH5a

  • Week 7/8

    • transform RFP & BL21(DE3)
    • PCR of pCas9, pTRC99A
    • Gibson – pick 8 colonies, make O/N colonies, try to PCR form reaction to get 4-fragment superchunk
    • Maraffini – use Anderson high for 4 fragment (phosphorylation + annealing of spacers if we get a lot of DNA), otherwise using Aug 18 miniprep do a BsaI digest
      • got colonies from Gibson
    • 3 things in parallel:
      • miniprepped plasmids from Gibson transformants from O/N liquid cultures (need to be sent for sequencing) , plasmids digested w E+P and to be run on gel
      • making competent MG1655 – transform RFP into it
      • tried BsaI digestion and gel extraction on Stanford-brown pCas9 (for Marafini/Golden-gate), does not seem to work even w large volumes
    • look at plates from transformation, run E+P digests on gel – if the digests look wrong size, do colony PCR on rest of Gibson colonies on plates
      • use fwd/rev primers for cas9 and p!TRC chunks
      • if correct, also PCR the 3-fragment superchunk from the miniprep plasmid by using Cas9 fwd with p!TRC rev primers
    • try a gel-extraction protocol…

  • Week 9

    • Main thing – GIBSON ASSEMBLY
    • screened 8 colonies – miniprepped, EcoRI + PstI digest & wrong product, non-specific assembly
      • extend gblocks (Anderson high + tracr) so more overlap, try Gibson again
    • did digests of plasmids w one restriction enzyme instead of double digest + run gel (for less confusing results) – used miniprep #1-4
    • made O/N cultures from first Gibson reaction #9-15 – miniprepped and streak out plates
    • minipreps #5-8 from earlier digested w EcoRI and PstO (separately), run on gel
    • take 2 minipreps, do 3 reactions on each – EcoRI only, PstI only, both EcoRI + PstI

  • Week 10

    • miniprep out RFP plasmid to be used for putting biobricks into pSB1C3, will want to screen for non-red colonies at the end of the process
    • unable to do digest because nanoprep of all 3 minipreps was too low
    • primers arrived – ran PCR w new primers
      • also digested RPF
      • run digests on gel (1/2 thickness)
    • ran the original gblocks w extended gblocks – not enough of original gblocks to show up on gel
    • combined PCR reactions of tracr gblocks into one, same with Anderson High PCR products
      • purified both
      • PCR purified Cas9 array from a PCR in August
      • purified p!TRC chunk found
      • all 4 to be used for Gibson assembly!
    • glycerol stocked 3 tubes of colonies from first Gibson attempt (#3, 4, 7)
    • transformed Gibson in NEB5alpha, ½ of each reaction tube into a total of 4 NEB5alpha – 4 tubes plated onto 2 plates each = total 8 plates incubated
    • on Friday, found colonies from the Gibson assembly and did colony PCR
    • colony PCR primers were designed to cover the seams where the 4 chunks joined
    • mixed the template DNA and biobrick primers for the Cas9-array chunk, but did not add the master mix yet (done later)

  • Week 11

    • ran Gibson colony PCR on gel – colonies #3 + 6 have correct bands, started O/N cultures of them w cell suspensions from colony PCR
    • miniprep and glycerol stock them (low copy plasmid – do 3uL miniprep)
    • PCR of cas9 chunk – run on gel, see if band is at 4595
    • using RFP miniprep from earlier, digest w EcoRI and PstI
      • linearised plasmid digested same way (if DNA dissolved – was left sitting overnight to dissolve)
    • nothing on gel at correct size…. only used 1uL of DNA for gel though
    • digest ~1000ng of Gibson miniprep from 2 colonies w EcoRI and PstI, run on gel
    • run 5uL of tube labelled C6*
    • cas9-array chunk biobrick extension PCR barely worked – not enough DNA after PCR purification but tracr chunk biobrick PCR worked
      • digested/ligated into pSB1C3, transformed into competent cells
      • Gibson colonly #3 didn’t assemble correctly, only produced 1 band from E+P digestion
    • transformed ligation into NEB5alpha – after 2 hour (actual = 10 min) incubation in SOB, pipetted into 2mL LB Cm and grew overnight
    • miniprepped to see if it grew and packed it up for shipping!

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