Team:Brown-Stanford/Lab/Notebook/Week12

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Latest revision as of 18:10, 28 September 2011

Brown-Stanford
iGEM
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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

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