Team:Brown-Stanford/Lab/Notebook/Week12

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== ''' August 29, 2011''' ==
-
== ''' August 22, 2011''' ==
+
*digestion
-
===PowerCell===
+
*Ana-TOPO
-
*Retransform LuxBrick into TOP10
+
*40 μl
-
*Digest positive controls:
+
*lux-pSB1C3
-
*EP, XS, ES, XP; run alongside uncut plasmids (in at 1245pm)
+
*40 μl
-
*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
+
*cscB PCR (linear)
 +
*20 μl
 +
*followed special measures for using EcoRI and SpeI with Buffer 2
 +
*2.5% enzymes by volume
 +
*but also maintaining a 10 unit: 1μg ratio of EcoRI to plasmid to avoid star activity
 +
*digestion success: verification gel has solid bands where expected
 +
*ligation
 +
*actually pulled off 10 μl reactions
 +
*included Xho1 enzyme to break up undesired lux component and hopefully increase chances of proper ligation pairings
 +
*also have enough digestion product remaining to do gel extraction if necessary
 +
*Transformed Promoterless-GFPmut3b from registry: Well 22I, Plate 1
 +
== ''' August 30, 2011''' ==
-
== ''' August 23, 2011''' ==
+
*e coli W competency & transformation w/ RFP
-
===PowerCell===
+
*transformation of Ana TOPO +lux/pSB1C3 ligation w/ XhoI
-
*the TOPO test with Ana may have worked, many small colonies visible on the 45 μl plates.  YAAY!
+
*transformation of cscB PCR +lux/pSB1C3 ligation w/ XhoI
-
*Pick several colonies to separate 3ml LB+amp liquid cultures
+
*redo ligations from 8/29 with HindIII instead of XhoI
-
*Performed TOPO ligation and transformation with cscB and gfp PCR products.
+
*turns out XhoI cuts pSB1C3
-
*No visible growth on never UVed ligation
+
*transform ligations redux
-
*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.
+
*warmed plates
-
*two divergent workflows - same as before, cutting out typhoon
+
*transformation of promoterless GFP from registry
-
*TOPO, for super concentrations.  for great success!
+
*all transformations (except GFP) performed on both TOP10 and K12
-
== ''' August 24, 2011''' ==
+
*new liquid cultures of pRL443 and pRL623+25\
-
===PowerCell===
+
*Made 3 amp plates. Running out of LB. hopefully we dont need more.
-
*verification PCR of the ana-TOPO liquid culture
+
== ''' August 31, 2011''' ==
-
*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
+
-
== ''' August 25, 2011''' ==
+
-
===PowerCell===
+
-
*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
+
-
== ''' August 26, 2011''' ==
+
-
===PowerCell===
+
-
*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
+
 +
*we have transformants all over the place!
 +
*Our TOP10 sucks compared to K12
 +
*Many colonies on the Ana-TOPO+lux/pSB1C3 HindIII K12 plate
 +
*three colonies on the cscB-PCR+lux/pSB1C3 HindIII K12 plate
 +
*one colony on the promoterless GFP TOP10 plate
 +
*a few colonies on the Ana-TOPO+lux/pSB1C3 XhoI K12 plate
 +
*a few colonies on the Ana-TOPO+lux/pSB1C3 XhoI TOP10 plate
 +
*Update Sept 1: 2 colonies cropped up on the Ana HindIII TOP10 plate!
 +
*verification PCR and gel of BioBrick transformation results
 +
*liq cultures transformations if verification looks good
 +
*verification looked... unconclusive, some things that *could* be what we want.
 +
*liquid cultured them, will miniprep, verify PCR / sequence.
 +
*AHK 2,4,5 ; CHK 2 ; GFPN ; AXT 3
 +
*cyano transformation, orgy part
 +
*agar stab ecoli W
 +
== ''' September 1, 2011''' ==
 +
 +
*made 1 liter LB
 +
*and BG11 (for cyano plates)
 +
*another ligation of cscB+pSB1C3(w/ lux)
 +
*to transform and get more candidate colonies
 +
*re-inoculate GFP
 +
*transfer cyanos to selective plates
 +
*2 colonies cropped up on the Ana HindIII TOP10 plate Biobrick transform plate from Aug 30! Put in the 4C in 335.
 +
*Miniprepped AHK2,4,5 ; CHK2 ; AXT3 BB candidates
 +
*Nanodrop results:
 +
*AHK2: 75 ng/ul
 +
*AHK4: 76 ng/ul
 +
*AHK5: 76ng/ul
 +
*CHK2: 186ng/ul
 +
*AXT3: 193 ng/ul
 +
*Sent the miniprepped BB cands. off for sequencing from Elim
 +
*Replated dried cyano slides/nitrocellulose control test from Aug 31 onto plain BG11 Plates
 +
*Slides - added 10ul BG11 over dried spot, scratched around, sucked up as much as pos, put on plate
 +
*nitrocellulose: just dropped on plate
 +
== ''' September 2, 2011''' ==
 +
 +
*colonies growing on cscB redux K12
 +
*confirmed now that TOP10 sucks, will be using K12 only from now on
 +
*liq cult of GFP looks good, ready to miniprep
 +
*completed miniprep; only 22 ng/μl but thatll do
 +
*AHK4 plasmid sequencing results came out beautifully
 +
*WE HAVE ANA PROMOTER BIOBRICK!!
 +
*AHK5 also promising but AHK4 looks like our prize. AHK5 is correct in the reverse direction, but bad read in the fwd direction. From gel they look similar, so we believe they’re both correct, but we’re only choosing AHK4 because we only need one and it’s perfect in both fwd and reverse.
 +
*CHK2 turned out to be the luxbrick re-ligated into psb1c3 even though we cut it up 4 times with HindIII! Interesting. Wonder how effective adding HindIII really is.
 +
*unknown what AXT3 is.
 +
*digestion and ligation of Ana-GFP construct
 +
*50 μl digestions
 +
*cut Ana-pSB1C3 w/ Spe1, Pst1
 +
*cut GFPN w/ Xba1, Pst1
 +
*ana ligation ended up at 15μl, but adjusted enzymes accordingly, and checked with kosuke about best approach
 +
*adjusted ligase and buffer amounts accordingly
 +
*3:1 insert to vector molar ratio
 +
*~50 ng vector (ana-pSB1C3 digest)
 +
*ligated at room temp for 2 hrs, -> -20 until Monday
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Latest revision as of 18:10, 28 September 2011

Brown-Stanford
iGEM
Back to
iGEM HQ

August 29, 2011

  • digestion
  • Ana-TOPO
  • 40 μl
  • lux-pSB1C3
  • 40 μl
  • cscB PCR (linear)
  • 20 μl
  • followed special measures for using EcoRI and SpeI with Buffer 2
  • 2.5% enzymes by volume
  • but also maintaining a 10 unit: 1μg ratio of EcoRI to plasmid to avoid star activity
  • digestion success: verification gel has solid bands where expected
  • ligation
  • actually pulled off 10 μl reactions
  • included Xho1 enzyme to break up undesired lux component and hopefully increase chances of proper ligation pairings
  • also have enough digestion product remaining to do gel extraction if necessary
  • Transformed Promoterless-GFPmut3b from registry: Well 22I, Plate 1

August 30, 2011

  • e coli W competency & transformation w/ RFP
  • transformation of Ana TOPO +lux/pSB1C3 ligation w/ XhoI
  • transformation of cscB PCR +lux/pSB1C3 ligation w/ XhoI
  • redo ligations from 8/29 with HindIII instead of XhoI
  • turns out XhoI cuts pSB1C3
  • transform ligations redux
  • warmed plates
  • transformation of promoterless GFP from registry
  • all transformations (except GFP) performed on both TOP10 and K12
  • new liquid cultures of pRL443 and pRL623+25\
  • Made 3 amp plates. Running out of LB. hopefully we dont need more.

August 31, 2011

  • we have transformants all over the place!
  • Our TOP10 sucks compared to K12
  • Many colonies on the Ana-TOPO+lux/pSB1C3 HindIII K12 plate
  • three colonies on the cscB-PCR+lux/pSB1C3 HindIII K12 plate
  • one colony on the promoterless GFP TOP10 plate
  • a few colonies on the Ana-TOPO+lux/pSB1C3 XhoI K12 plate
  • a few colonies on the Ana-TOPO+lux/pSB1C3 XhoI TOP10 plate
  • Update Sept 1: 2 colonies cropped up on the Ana HindIII TOP10 plate!
  • verification PCR and gel of BioBrick transformation results
  • liq cultures transformations if verification looks good
  • verification looked... unconclusive, some things that *could* be what we want.
  • liquid cultured them, will miniprep, verify PCR / sequence.
  • AHK 2,4,5 ; CHK 2 ; GFPN ; AXT 3
  • cyano transformation, orgy part
  • agar stab ecoli W

September 1, 2011

  • made 1 liter LB
  • and BG11 (for cyano plates)
  • another ligation of cscB+pSB1C3(w/ lux)
  • to transform and get more candidate colonies
  • re-inoculate GFP
  • transfer cyanos to selective plates
  • 2 colonies cropped up on the Ana HindIII TOP10 plate Biobrick transform plate from Aug 30! Put in the 4C in 335.
  • Miniprepped AHK2,4,5 ; CHK2 ; AXT3 BB candidates
  • Nanodrop results:
  • AHK2: 75 ng/ul
  • AHK4: 76 ng/ul
  • AHK5: 76ng/ul
  • CHK2: 186ng/ul
  • AXT3: 193 ng/ul
  • Sent the miniprepped BB cands. off for sequencing from Elim
  • Replated dried cyano slides/nitrocellulose control test from Aug 31 onto plain BG11 Plates
  • Slides - added 10ul BG11 over dried spot, scratched around, sucked up as much as pos, put on plate
  • nitrocellulose: just dropped on plate

September 2, 2011

  • colonies growing on cscB redux K12
  • confirmed now that TOP10 sucks, will be using K12 only from now on
  • liq cult of GFP looks good, ready to miniprep
  • completed miniprep; only 22 ng/μl but thatll do
  • AHK4 plasmid sequencing results came out beautifully
  • WE HAVE ANA PROMOTER BIOBRICK!!
  • AHK5 also promising but AHK4 looks like our prize. AHK5 is correct in the reverse direction, but bad read in the fwd direction. From gel they look similar, so we believe they’re both correct, but we’re only choosing AHK4 because we only need one and it’s perfect in both fwd and reverse.
  • CHK2 turned out to be the luxbrick re-ligated into psb1c3 even though we cut it up 4 times with HindIII! Interesting. Wonder how effective adding HindIII really is.
  • unknown what AXT3 is.
  • digestion and ligation of Ana-GFP construct
  • 50 μl digestions
  • cut Ana-pSB1C3 w/ Spe1, Pst1
  • cut GFPN w/ Xba1, Pst1
  • ana ligation ended up at 15μl, but adjusted enzymes accordingly, and checked with kosuke about best approach
  • adjusted ligase and buffer amounts accordingly
  • 3:1 insert to vector molar ratio
  • ~50 ng vector (ana-pSB1C3 digest)
  • ligated at room temp for 2 hrs, -> -20 until Monday

Retrieved from "http://2011.igem.org/Team:Brown-Stanford/Lab/Notebook/Week12"