Team:Toronto/Notebook

From 2014.igem.org

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<ul>
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<li class="week">
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<br>
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<h2><span>Week 1</span></h2>
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<ul class="thingsdone">
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<li>
 +
picked single MG1655 and pTRC99A strains into liquid culture,
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incubated overnight (pTRC99A in LB+Amp)
 +
</li>
 +
<ul>
 +
<li>
 +
miniprepped pTRC, O/N culture of MG1655
 +
</li>
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<li>
 +
MG1655 did not grow on antibiotic plates, so stocks are good!
 +
</li>
 +
</ul>
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<li>
 +
prepped DNA for transformation – pCas9 and RFP reporter
 +
</li>
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<li>
 +
made MG1655 electrocompetent –
 +
electroporate and plated transformants, incubated overnight
 +
</li>
 +
</ul>
 +
</li>
 +
<li class="week">
 +
<h2><span>Week 2</span></h2>
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<ul class="thingsdone">
 +
<li>
 +
transformation of RFP, pCas9, GFP, fmt into NEB5alpha
 +
</li>
 +
<ul>
 +
<li>
 +
transformants plated and incubated overnight
 +
</li>
 +
<li>
 +
then inoculated into liquid culture, incubated overnight
 +
</li>
 +
</ul>
 +
<li>
 +
overnight culture of MG1655 and pTRC99A
 +
</li>
 +
<li>
 +
Miniprepped RFP and pTRC99A
 +
</li>
 +
<ul>
 +
<li>tested restriction enzymes EcoRI, Xbal, SpeI, Pstl
 +
on miniprep and ran on gel w/ uncut as control
 +
</li>
 +
<li>
 +
cut plasmids were too faint to see – need more
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concentrated DNA in preps!
 +
</li>
 +
</ul>
 +
<li>
 +
O/N cultures of RFP, pCas9, GFP from transformation
 +
</li>
 +
<li>
 +
miniprep of O/N cultures of transformants and pTRC99A
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</li>
 +
</ul>
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<li class="week">
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<h2><span>Week 3</span></h2>
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<ul class="thingsdone">
 +
<li>
 +
miniprep again, greater volume (3mL)
 +
for RFP, GFP, Cas9, pTRC99A + nanodrop
 +
</li>
 +
<ul>
 +
<li>
 +
digest product (w SpeI, EcoRI, cutsmart) and run on gel
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</li>
 +
</ul>
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<li>
 +
O/N culture from plate of extra biobrick, RFP, MG1655
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</li>
 +
<li>
 +
start RFP and MG1655 liquid cultures for the RFP
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spectrum measurements in the afternoon
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</li>
 +
<ul>
 +
<li>
 +
RFP grew slowly and was transformed into NEB5alpha instead of MG1655
 +
</li>
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</ul>
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<li>
 +
digest 8 samples from yesterday’s miniprep of 8 biobrick cultures,
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load and run gel of digest
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</li>
 +
<li>
 +
oligos and gblocks arrived
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</li>
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<li>
 +
did PCR of pCas9 and pTRC99A chunks
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</li>
 +
<li>
 +
Started MG1655 liquid culture for making chemically competent
 +
</li>
 +
<ul>
 +
<li>
 +
make chemically competent cells, transform RFP into them,
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PCR purification, run on gel
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</li>
 +
</ul>
 +
</ul>
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<li class="week">
 +
<h2><span>Week 4</span></h2>
 +
<ul class="thingsdone">
 +
<li>
 +
miniprepped pCas9 + pTRC99A (w 7mL of cells)
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</li>
 +
<li>
 +
did gel extraction – cut out pCas9 + pTRC digests –
 +
PCR both as well as nanodrop
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</li>
 +
<li>
 +
Mon/Tues = PCR w mastermix,
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Wed = make comp MG1655 and heatshock RFP,
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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>
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</ul>
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<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
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</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
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</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
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plasmid but very few colonies on CM plate
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</li>
 +
</ul>
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<li class="week">
 +
<h2><span>Week 6</span></h2>
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<ul class="thingsdone">
 +
<li>
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PCR product (???) run on gel, pick most successful PCR
 +
</li>
 +
<li>
 +
DpnI digest following protocol in NEB Gibson manual
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Transform RFP & MG1655, O/N cultures
 +
</li>
 +
<li>
 +
pCas9PCR product visible on gel
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</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
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</li>
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</ul>
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<li class="week">
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<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>
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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>
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<li class="week">
 +
<h2><span>Week 9</span></h2>
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<ul class="thingsdone">
 +
<li>
 +
Main thing – <b>GIBSON ASSEMBLY</b>
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</li>
 +
<li>
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screened 8 colonies – miniprepped, EcoRI + PstI digest & wrong product, non-specific assembly
 +
</li>
 +
<ul>
 +
<li>
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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
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</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
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</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,  
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same with Anderson High PCR products
 +
</li>
 +
<ul>
 +
<li>
 +
purified both
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</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>
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glycerol stocked 3 tubes of colonies from first
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Gibson attempt (#3, 4, 7)
 +
</li>
 +
<li>
 +
transformed Gibson in NEB5alpha, ½ of each reaction
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tube into a total of 4 NEB5alpha – 4 tubes plated
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onto 2 plates each = total 8 plates incubated
 +
</li>
 +
<li>
 +
on Friday, found colonies from the Gibson assembly and
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did colony PCR
 +
</li>
 +
<li>
 +
colony PCR primers were designed to cover the seams
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where the 4 chunks joined
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</li>
 +
<li>
 +
mixed the template DNA and biobrick primers for the
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Cas9-array chunk, but did not add the master mix yet
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(done later)
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</li>
 +
</ul>
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<li class="week">
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<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
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</li>
 +
<li>
 +
miniprep and glycerol stock them (low copy plasmid – do 3uL miniprep)
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</li>
 +
<li>
 +
PCR of cas9 chunk – run on gel, see if band is at 4595
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</li>
 +
<li>
 +
using RFP miniprep from earlier, digest w EcoRI and PstI
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</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
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</li>
 +
<li>
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digest ~1000ng of Gibson miniprep from 2 colonies w EcoRI and PstI, run on gel
 +
</li>
 +
<li>
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run 5uL of tube labelled C6*
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</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
 +
</li>
 +
</ul>
 +
<li>
 +
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>
 +
<li>
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miniprepped to see if it grew and packed it up for shipping!
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Pro posse lobortis an. Ea has paulo definiebas. Usu et doctus mentitum. Ea mea iisque accumsan, paulo utroque reprimique ea per, agam erant pri ex. Quodsi timeam perfecto mel ei, ius ex odio tamquam quaeque. Eruditi fabellas eum ut, per et illud propriae, eu error utroque mnesarchum mei.
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Pro posse lobortis an. Ea has paulo definiebas. Usu et doctus mentitum. Ea mea iisque accumsan, paulo utroque reprimique ea per, agam erant pri ex. Quodsi timeam perfecto mel ei, ius ex odio tamquam quaeque. Eruditi fabellas eum ut, per et illud propriae, eu error utroque mnesarchum mei.
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Per ut impetus sententiae, fuisset constituto eos id. Illud definitionem vis ea, affert graeco mnesarchum at nec, has ex ponderum cotidieque. Et eos decore vidisse deseruisse, erat ornatus vivendo et eam. Semper audiam ne pro. Quot impedit per et, ad justo aperiam omnesque pri. Sonet noluisse assueverit ius in.
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Ad causae feugait accusata pro, usu amet tale laoreet ad. Debet simul ponderum vim in. Sed te graecis deleniti, ex per nominati urbanitas. At decore tamquam vel. Vis in illum error honestatis, eam tantas audire adversarium id. Ius ut eloquentiam signiferumque. Ei nec partem scripserit, solet invidunt at nam, nec in minimum eligendi delicatissimi.
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Tempor cotidieque quo at, dolores nominavi abhorreant ius eu. At pro illum instructior. At enim aperiam honestatis quo. Ad vis ornatus offendit voluptatibus. Ad eruditi feugait inciderint sea, solet denique cu eos.
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Pro posse lobortis an. Ea has paulo definiebas. Usu et doctus mentitum. Ea mea iisque accumsan, paulo utroque reprimique ea per, agam erant pri ex. Quodsi timeam perfecto mel ei, ius ex odio tamquam quaeque. Eruditi fabellas eum ut, per et illud propriae, eu error utroque mnesarchum mei.
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Per ut impetus sententiae, fuisset constituto eos id. Illud definitionem vis ea, affert graeco mnesarchum at nec, has ex ponderum cotidieque. Et eos decore vidisse deseruisse, erat ornatus vivendo et eam. Semper audiam ne pro. Quot impedit per et, ad justo aperiam omnesque pri. Sonet noluisse assueverit ius in.
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<h2><span>Heading 2</span></h2>
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Lorem ipsum dolor sit amet, mel at reprehendunt comprehensam, erant iusto exerci at usu. Usu ornatus rationibus cu, sed veritus minimum te, quas novum percipitur ut vis. Ea mei accusamus disputando, qui eros duis aliquando cu. Dicunt mandamus reprimique te sea. Tation hendrerit sea eu, ut harum aliquid vix.
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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!

Retrieved from "http://2014.igem.org/Team:Toronto/Notebook"