Team:Brown-Stanford/Lab/Notebook/Week11

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Jamboree

August 22, 2011

Retransform LuxBrick into TOP10
Digest positive controls:
EP, XS, ES, XP; run alongside uncut plasmids (in at 1245pm)
positive control bands showed up at significantly higher sizes than expected? otherwise, undig controls showed 3 bands (corresponding to three plasmid conformations), digested ones all showed two; same with lux brick

the TOPO test with Ana may have worked, many small colonies visible on the 45 μl plates. YAAY!
Pick several colonies to separate 3ml LB+amp liquid cultures
Performed TOPO ligation and transformation with cscB and gfp PCR products.
No visible growth on never UVed ligation
New Idea! What could be destroying our ligations/transformations is the UV from the TYPHOON! We forgot about this. It blasts at very high intensity UV for three minutes. Come to think of it, none of our transformations that have gone though the typhoon have ever worked, we think. Use just transluminator. Also, goddamn.
two divergent workflows - same as before, cutting out typhoon
TOPO, for super concentrations. for great success!

verification PCR of the ana-TOPO liquid culture
success!
PCR amplification of pSB1C3
fail
liquid cultures of gfp and cscB
miniprep of lux brick cultures
2 preps of 10 mL tubes
result: 590 ng/μl and 600 ng/μl
can use this to digest out backbone for other ligation reactions because PCR of backbone to use with TOPO didn’t work
Inoculated big 10ml Ana TOPO liq cults.
All were innoculated from Ana TOPO LC #1
Cyrostocked Ana TOPOs
cyrostocked all 4 LC’s
50% glycerol - couldn’t find 40%, was in a hurry.
Put in brown cryobox with RegoBricks stuff in -80

verification PCR of GFP and cscB TOPO clones
ran gel, positive control worked, bu not bands in any other lanes
we think that since we didn’t shake the liquid culture templates, the cells settled and we didnt get enough cells.
miniprep of Ana TOPO liq cultures(4, 10mL each)
91 ng/μl, 165ng/μl, 173 ng/μl, 182 ng/μl

run gel to verify GFP & cscB TOPO PCR
2nd fail
no bands where desired
seem to be bands down around where the empty vector would show up but hard to say because all we have is 500bp ladder
don’t have money to order ladder, etc waiting on Brown iGEM funding approval
lab meeting