Team:Arizona State/Notebook

From 2012.igem.org

(Difference between revisions)
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* Plated BBa_K283010 (Streptavidin) on LB amp plate from the agar stab provided from the iGEM head-quarters.
* Plated BBa_K283010 (Streptavidin) on LB amp plate from the agar stab provided from the iGEM head-quarters.
* Incubated the dish overnight at 37 C.
* Incubated the dish overnight at 37 C.
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[[Image:ASUiGEM2012_plates072312.jpeg|200px]]
+
[[File:ASUiGEM2012_plates072312.jpeg|200px]]
==July 24==
==July 24==

Revision as of 05:46, 3 October 2012


June 2012
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July 2012
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August 2012
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September 2012
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October 2012
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June 07

  • Transformation (LSE)
    • Transformed DNA:
      • lacZ (well 4:12G, I732019)
      • p + lacO (well 1:6G, R0011)
    • Cells: neb10beta (donated)
    • Protocol from: http://www.neb.com/nebecomm/products/productc3019.asp
    • Controls: puc19, no DNA (8 plates)

June 08

  • Transformation results
    • puc19: growth
    • negative control: no growth
    • lacZ, lacO: possible small colonies
  • liquid culture in amp media (100 ug / ml):
    • no growth of lacZ, lacO
    • growth of puc19

June 12

  • DH5a Competent Cell Prep
    • Streak plated cells on LB no amp plate, let grow overnight

June 13

  • Transformation (LSE)
    • Transformed DNA:
      • lacZ (well 4:12G, I732019)
      • p + lacO (well 1:6G, R0011)
    • Cells: DH5 alpha (donated)
    • Protocol from: http://openwetware.org/wiki/Haynes:Assembly101 (30 minute transformation)
    • Controls: puc19, no DNA (8 plates)
  • Transformation (istb4, Abhi)
    • Transformed DNA:
    • Cells:
    • Protocol from:
    • Controls:
  • DH5a Chemically Competent cell prep
    • Grew 2 seed colonies from streak plate in LB no amp
    • Grew controls to test for contamination
      • Both Seed colonies grew, no contamination present

June 14

  • Competent cell prep
    • Prepared CaCl2 buffer solution and CaCl2 glycerol buffer solution
    • Grew seed colony in 400mL LB no amp

June 15

  • Competent cell prep
    • Centrifuged falcon test tubes containing liquid colonies
    • Resuspended in CaCl2 buffer solution and incubated for 15 mins
    • Centrifuged and resuspended in CaCl2 glycerol buffer solution
    • Chilled overnight

June 16

  • Competent cell prep
    • Aliquotted 200uL into test tubes
    • Stored in -80C

June 17

  • Streak plated prepared competent cells on LB no amp plate
    • Colonies observed

June 19

  • Transformation (LSE)
    • Transformed DNA:
      • T7 promoter BBa_I712074
      • Constitutive promoter BBa_J23102
    • Cells: DH5 alpha (donated)
    • Protocol from: http://tools.invitrogen.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf (30 minute transformation)
    • Controls: puc19, no DNA
    • Plated 2 copies of each (100 ul, 250 ul) on LB amp plates.
  • Made 50 LB Amp plates.

June 20

  • Plated negative control on LB Amp plate
  • Liquid cultures of T7 promoter and constitutive promoter
  • Transformation (LSE)
    • Transformed DNA:
      • RBS (well 1:1H BBa_B0030)
      • TetR GFP (well 2:8A Part:BBa_I13522)
    • Cells: DH5 alpha (donated)
    • Protocol from: http://tools.invitrogen.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf (30 minute transformation)
    • Controls: puc19, no DNA

June 21

  • Made Liquid Cultures of E.coli transformed with RBS B0030
  • Made Liquid Cultures of E.coli transformed with TetR GFP
  • miniprepped and nanodropped T7 promoter BBa_I712074 and Constitutive promoter BBa_J23102 liquid cultures
  • liquid cultures:
    • RBS1
    • RBS2 (duplicate_
    • GFP1
    • puc19
    • negative controls
    • 5 ml LB amp
    • overnight cultures
  • replated GFP1 & 2 (duplicates)
  • Nanodropped plasmid DNA samples
    • Constitutive promoter 1: __ng/uL
    • Constitutive promoter 2: __ng/uL
    • T7 promoter 1: __ng/uL
    • T7 promoter 2: __ng/uL

June 22

  • Miniprepped liquid cultures: RBS (well 1:1H BBa_B0030) and TetR GFP (well 2:8A Part:BBa_I13522)
  • Picked colonies:
    • 1 colony from double terminator (dt1) plate
    • 1 colony from t7 polymerase (pol1) plate
    • 1 colony from puc19 plate (positive control)
    • 1 colony from dh5a plate (negative control)
  • started liquid cultures of each colony (5 mL LB amp each)

June 26

  • Transformation:
    • Transformed DNA:
      • double terminator (B0017, 2:6K)
      • T7 RNA polymerase (I715038, 2:15C)
      • puc19, negative control
    • Protocol from: http://tools.invitrogen.com/content/sfs/manuals/subcloningefficiencydh5alpha_man.pdf (30 minute transformation)
    • Cells: dh5a

June 27

  • 6-26 transformation results:
    • Controls correct
    • 2x terminator: ~19 colonies
    • RNA pol: 1 colony
  • Liquid cultures including controls

June 28

  • Miniprepped double terminator (B0017, 2:6K) and T7 RNA polymerase (I715038, 2:15C) liquid cultures

July 2

  • Cleaned up liquid waste
  • Made SOB media
  • Finalized oligos for magainin construct

July 3

  • Autoclaved SOB media
  • Added glucose to make SOC media
  • Nanodropped double terminator (B0017, 2:6K) [DT1: 24.5, DT2: 29.6] and T7 RNA polymerase (I715038, 2:15C) [P1: 64.6, P2: 55.3] liquid cultures

July 15

  • Ran a miniprep of Bgal alpha 1&2, T7 and PSV
  • Procedure from GenElute HP PLasmid Miniprep Kit (Sigma-Aldrich)

July 16

  • Transformation of PLflex (part: BBa_J176040) and PLflex4 (part: BBa_J176130) linkers.
  • Incubate at 37C for 8 hours for DH 5alpha turbo cells
  • Inoculated 5 mL LB/AMP media in 15 mL culture tubes and added the colony. Incubated in 37C overnight.

July 17

  • Miniprep of pLFlex DH5alpha and pLFlex 4 DH5alpha
  • Procedure comes from Zymo Research's Zyppy Kit.
  • Ran the spectrophotometer to determine the concentration for the miniprep DNA for the linker pLFlex and linker pLFlex 4
  • Concentrations were:
    • pLFlex: 73.792 ng/uL
    • pLFlex 4: 38.587 ng/uL
  • Absorbance ratios were:
    • pLFlex: 1.864
    • pLFlex 4: 1.851
  • Ran the miniprep for pLFlex and pLFlex 4 again.
  • Same procedure
  • Ran the spectrophotometer again
  • Concentrations were:
    • pLFlex: 48.095 ng/uL
    • pLFlex 4: 18.214 ng/uL
  • Absorbance ratios were:
    • pLFlex: 1.79
    • pLFlex 4: 1.85
  • Transformation was successful but 8-9 hour growth period was not sufficient for DH5 alpha.

July 23

  • Plated BBa_K283010 (Streptavidin) on LB amp plate from the agar stab provided from the iGEM head-quarters.
  • Incubated the dish overnight at 37 C.

200px

July 24

  • Plasmids arrived courtesy of University of Pennsylvania School of Medicine
  • pET29a vectors containing coding sequence for Topoisomerase mutants CSCS and CSCS D168A described here

July 25

  • Tranformed CSCS topo 0 plasmid and CSCS D168A topo into DH5a Turbo cells (with neg control and Puc19 neg control)
  • Plated on Kanamycin plates

July 26

  • Picked colonies colonies from Topo 0 and Topo D168A and grew liquid cultures in Kan media

July 27

  • Miniprepped liquid colonies and nanodropped.
  • Plasmid concentrations
    • Topo O:
    • Topo D168A1:
    • Topo D168A2:

July 30

  • Prepared Kan Media and Kan Plates

July 31

  • PCR amplified polylinker sequence of Topo plasmid with Promega GoTaq protocol
    • Used pET29a upstream forward primer and T7 terminator reverse primer

August 3

  • Submitted pET29a Topoisomerase plasmid to Biodesign for sequencing
  • Resuspended GFPT1 and GFPT2 oligos with molecular grade (nuclease-free) H2O.
    • Final Concentration 100uM
      • (gfpt1 top1, gfpt2 top1, gfpt1 top2, gftp2 top2, gfpt1 bot1, gfpt2 bot1, gfpt1 bot2, gfpt2 bot2)
      • (3uL of each oligo + 2uL 10x annealing buffer, 6uL molecular grade H2O. 20uL Reactions)
  • Heated for 5 minutes at 100C. Let cool to room temperature on the heating block, stored at -20C.
  • Digested BBa_I13522 with XbaI and PstI.
  • Attempted ligating annealed oligos into a digested plasmid from Ryan (realized it was cut with E and P).

August 6

  • Annealed oligos for GFPT1 and GFPT2 (target probes)
  • Ligated oligos with digested GFP plasmid (BBa_I13522)
  • Transformed into competent DH5alpha
  • Added SOC and incubated at 37C for 15 minutes.
  • Plated on amp treated plates.

August 7

  • Only one colony on each plate (both were white)
  • Picked colonies and started 5mL LB amp cultures of each, stored at 37C
  • Stored plates in 37C

August 8

  • Picked the colonies again and started new liquid cultures (5mL LB amp).
  • Discarded cultures for 8/7/12

August 9

  • Miniprepped 3mL of each 8/8/12 culture and nanodropped:
    • gfpt1 - 155 ng/uL
    • gfpt2 - 114 ng/uL
  • Digested gfpt1 and gfpt2 with X and P
  • Ran on a 1% agarose gel with the digested GFP plasmid
  • Made glyercol stocks with aliquot of the remaining liquid cultures

August 10

  • PCR of Alpha-4, 1-omega, omega, and alpha fragments using corrected primers

August 13

  • Ran gel of split beta gal fragments. Confirmed 3 out of the 4 fragments except for the alpha-4 fragment.

ASUiGEM2012 gel081312.jpeg

  • Prepared sequencing samples
  • Sample w/ Primer:
    • GFPT1 w/ VF2 GFPT1 w/ VR GFPT2 w/ VF2 GFPT2 w/ VR
    • 200ng of DNA + 16 pmol of primer
  • Annealed oligos again GFPT1/2
  • Repeated ligation of oligos with digested GFP plasmid (BBa_I13522)
  • Followed Haynes assembly protocol instead of standard DH5alpha protocol. (http://openwetware.org/wiki/Haynes:Assembly101)
  • Transformed ligations into competent DH5alpha
  • Plated on amp treated plates

August 14

  • Took pictures of plates
  • Green-white screened plates
  • Picked 4 white colonies from each of gfpt1/2 plates
  • Made 5mL LB amp cultures of each colony
  • Delivered GFPT1/2 dna samples to biodesign for sequencing (samples from 8/13/12)
  • Assembled magainin insert via Overlapping oligo assembly
  • Digested pUC 19 plasmid with EcoRI and PstI
  • Transformed Magainin insert into digested pUC 19 plasmid. Failed. Probably too much X-gal on plate.

Ran gel for the beta gal alpha-4 fragment. Failed. Fragment not in the correct size-band.
ASUiGEM2012 gel081412.jpeg

August 15

  • [http://129.219.2.10/home2/dnalims/fragment/32/21/pfasta/topo0_T7Term.seq Topo 0], [http://129.219.2.10/home2/dnalims/fragment/32/21/pfasta/D168Atopo1_T7Term.seq D168A Topo1], and [http://129.219.2.10/home2/dnalims/fragment/32/21/pfasta/D168Atopo2_T7Term.seq D168A Topo2] sequence results
  • Miniprepped 3mL of each liquid culture of GFPT1/2
  • Prepared glycerol stocks using 100uL of each liquid culture
  • Nanodropped samples:
    • GFPT1-1 - 172.6 ng/uL
    • GFPT1-2 - 203.7 ng/uL
    • GFPT1-3 - 197.4 ng/uL
    • GFPT1-4 - 178.9 ng/uL
    • GFPT2-1 - 107.3 ng/uL
    • GFPT2-2 - 131.2 ng/uL
    • GFPT2-3 - 145.5 ng/uL
    • GFPT2-4 - 172.0 ng/uL

August 16

  • Replated magainin insert + plasmid into the grid. Failed. All blue colonies meaning that no insert.

Tried gel for all gel-isolated fragments. Failed. Did not get a band in the 2000 bp region. Only got things below 200 bp.
ASUiGEM2012 gel081612.jpeg

August 17

  • GFPT1 sequence confirmed
  • Prepared aliquots of GFPT2 minipreps from 8/15/12 for sequencing
  • Delivered GFPT2 samples to biodesign for sequencing

August 19

  • GFPT2 sequences confirmed

August 27

  • Revived GFPT1 (from 8/9/12) and GFPT2 (2-2 from 8/15/12) cultures from glycerol scrapes
  • Made 4mL cultures in LB Amp

August 29

  • Discarded GFPT1/2 cultures from 8/27/12
  • Revived GFPT1 (from 8/9/12) and GFPT2 (2-2 from 8/15/12) cultures from glycerol scrapes
  • Made 4mL cultures in LB Amp
  • Digested GFPT1/2 with X and S
  • Ran a 1% agarose gel with GFPT1/2 digestions
  • Cut out inserts and GFPT2 backbone and stored in 4C for gel extraction and tandum repeat assembly experiments

August 30

  • Prepared extra glyercol stocks of GFPT1/2 cultures from 8/29/12
  • Miniprepped 3mL of each culture, stored at -20C

September 19

  • Set up VF2/VR endpoint PCR for double transform minipreps
    • 1-1, 1-1I, 1-2, 1-2I, 1-3, 1-3I, 2-1, 2-1I, 2-2, 2-2I, 2-3, 2-3I, GFPT1 (positive controls), GFPT2 (positive controls)
  • Annealing temp set to 55C for 25 cycles
  • Resuspended GFPT1 probe and GFPT2 probe oligos in molecular grade H2O (Final concentration: 100uM), stored at -20C

September 25

  • Resuspended pSB1A2 FWD and pSB1A2 REV (amp resistance primers) oligos in molecular grade H2O (Final concentration: 100uM), stored at -20C
  • Prepared 1.6uM dilutions (500uL)
  • Did endpoint PCR using pSB1A2 primer pair on
    • Topo, Topo IPTG, Topo D168A, Topo D168A IPTG, 1-1, 1-1I, 2-1, 2-1I, GFPT1, GFPT2
  • Did endpoint PCR using VF2/VR primer pair on
    • Topo, Topo IPTG, Topo D168A, Topo D168A IPTG
  • Annealing temp 55C for 25 cycles, stored products at -20C
  • Made 3mL liquid cultures of the shipping vector (pSB1C3 with RFP insert) in DH5alpha in chloramphenicol resistant LB
  • Made 10mL liquid cultures of:
    • Topo in kanamycin
    • topo D168A in kanamycin
    • topo + GFPT1 in kanamycin + ampicillin
    • topo + GFPT2 in kanamycin + ampicillin
  • stored @ 37C

September 26

  • Picked two new colonies for topo + GFPT2 and grew 10mL cultures of amp+kan LB broth for each
  • Ran a gel with:
    • Hyperladder I, GFPT1 pSB1A2, GFPT2 pSB1A2, Topo pSB1A2, Topo D168A
  • Ran a gel with PCR samples from 9/19 and 9/25:
    • Hyperladder I, 1-1, 1-1I, 2-1, 2-1I, 2-1I(VF), 2-1(VF), 1-1I(VF), 1-1(VF), Hyperladder I
  • Samples in wells 2-5 used the pSB1A2 primer pair. Samples in wells 6-9 used VF2/VR primer pair.
  • Revived GFPT1/2 from glycerol stocks (from 8/9 and 8/15) in 5mL LB amp each.
  • Nanodropped miniprepped DNA samples:
    • pSB1C3 I - 253.75 ng/uL
    • pSB1C3 II - 258.38 ng/uL
    • Topo - 24.84 ng/uL
    • Topo I - 17.33 ng/uL
    • Topo D168A - 7.24 ng/uL
    • Topo D168A I - 15.62 ng/uL
    • 1-1 - 16.49 ng/uL
    • 1-1I - 16.46 ng/uL
    • 2-1 - 142.05 ng/uL
    • 2-1I - 16.98 ng/uL
  • Picked new colonies from Topo, Topo D168A, Topo + G1, Topo + G2 and made 1mL colonies in their respective medias
  • Stored at -37C

September 27

  • Prepared serial dilutions of GFPT2 plasmid 1:10, 1:100, 1:1000, 1:10000
  • Prepared primer mixes for pSB and VF/VR primers
  • Prepared realtime PCR plate, ran RT-qPCR with annealing temp 57
  • Miniprepped GFPT1/2 cultures from 9/26
  • Nanodropped Miniprepped DNA:
    • GFPT1 - 276.12 ng/uL
    • GFPT2 - 219.84 ng/uL
  • Used 100uL of each 1mL culture from 9/26 to seed 10 mL cultures in their respective media
  • Added 10uL of 1M IPTG to each culture ~4 hours after seeding
  • Removed cells from 37C ~4 hours after IPTG inducing
  • Pelleted and lysed following the bugbuster protocol (http://openwetware.org/wiki/User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli/2012/05/22) (used lysonase for Topo and Topo D168A only)
  • HIS purified proteins using the Zymo HIS purification kit
  • Digested pSB1C3 plasmid with X and P
  • Sent protein samples (HIS purified samples from 9/19, 20uL) and ssDNA control (GFPT1/2 probe oligos 20uL @ 10uM) for mass spec

September 28

  • Resuspended 8 new oligos, final volume 100 uM each
  • Annealed pet29 top/bot oligos
  • Redid the RT-PCR, 3x primer concentration, added 1:1 plasmid concentration
  • Made 1.6uM aliquots of primer stocks 1-7 (including topo add X primer previously ordered)
  • Diluted aliquot of Topo D168A plasmid 4:10
  • Diluted PSV plasmid 2:20
  • Performed Endpoint PCR on:
    • Topo D168A using primers 1,2 and primers 1,3
  • PSV using primers 4-5 and primers 6-7
  • 4 samples with low primer concentration, 4 samples with twice as much primer (labelled 'H')
  • Miniprepped Topo1 2, Topo1 3, Topo1 4 (biobricked Topo D168A without T7, multiple colonies from ligation into shipping vector)
  • Nanodropped:
    • Topo1 2 - 63.63 ng/uL
    • Topo1 3 - 75.07 ng/uL
    • Topo1 4 - 184.93 ng/uL
  • 1mL chloramphenicol cultures of GFPT1/GFPT2 in shipping vector prepared
  • Digested Topo1 2, Topo1 3, Topo1 4 with E and P
  • Ran digested Topo plasmids on 1% agarose gel with Hyperladder I
  • Revived cultures of 2xGFPT1, 2xGFPT2, Topo, and Topo D168A (4 mL cultures each in their respective medias)
  • T5 exonuclease treated miniprepped plasmids (1-1, 1-1I)
  • A1 1-1I + t5
  • B1 1-1 + t5
  • A2 1-1I untreated
  • B2 1-1 untreated

September 29

  • Miniprepped GFPT1, GFPT2, Topo, and Topo D168A (Topo cultures separated into 3mL and 1mL minipreps)
  • Nanodropped miniprepped DNA:
    • GFPT1 I - 275.29 ng/uL
    • GFPT1 II - 101.08 ng/uL
    • GFPT2 I - 222.11 ng/uL
    • GFPT2 II - 230.52 ng/uL
    • Topo I - 117.97 ng/uL
    • Topo II - 70.28 ng/uL
    • Topo D168A I - 69.47 ng/uL
    • Topo D168A II - 51.87 ng/uL
  • HIS purified crude lysates from 9/27/12 (Topo, Topo D168A, Topo + GFPT1, Topo + GFPT2, all IPTG induced)
  • Digested Topo I plasmid (pet29a) with E and X
  • Ran a gel with Hyperladder I, 1-2 1-3 4-5 and 6-7 PCR products, pet29a digestion, and pSB1C3 digestion
  • Confirmed that PCR made amplicons
  • Excised bands for digested pet29a and pSB1C3 plasmids
  • Gel extracted 4 gel fragments (2 wells per sample: digested pSB1C3 plasmid, digested pet29a plasmid)
  • Nanodropped gel extractions:
    • Digested pSB1C3 - 25.54 ng/uL
    • Digested pet29a - 22.95 ng/uL
  • Set up a bradford assay of topo, topo D168A, topo + G1, topo + G2 (5uL protein, 10uL protein, 20uL protein + 200uL reagent)
  • Used ~100uL aliquot of BL21 competent glycerol stock to seed 10mL of LB medium (no antibiotic), stored at 37C
  • Ran 1% agarose gel with samples: A1, B1, A2, B2 and Hyperladder I
  • Did bug buster protocol to lyse BL21 control culture (used lysonase)
  • Ligated digested pet29a with pet29 top/bot annealed oligos
  • Transformed ligation using invitrogen DH5alpha transformation protocol
  • Prepared LB kanamycin plates
  • plated transformed ligations on prewarmed kanamycin plates

September 30

  • Pet29a plates did not grow
  • Kinase treated pet29a oligos
  • Annealed kinase treated pet29a oligos
  • Ligated digested pet29a (from gel extraction) with kinase treated oligos
  • Transformed ligations into DH5alpha, used topo plasmid as a positive control
  • Plated transformations on kanamycin plates and stored overnight at 37C
  • HIS purified BL21 control crude lysate
  • Set up a bradford assay with:
    • uninduced & induced topo protein extractions from 9/19
    • uninduced & induced topo D168A protein extractions from 9/19
    • BL21 control lysate
    • Topo, Topo D168A, Topo + G1, Topo + G2 from 9/27
  • All samples prepared (10uL protein, 20uL protein + 200uL reagent)
  • Miniprepped GFPT1 1,2,3 and GFPT2 (17,18,26) (~600uL of each) (1mL liquid cultures made from colonies on the chloramphenicol plates of GFPT1 and GFPT2 ligated into the shipping vector)
  • Nanodropped:
    • GFPT1-1 - 38.5 ng/uL
    • GFPT1-2 - 77.6 ng/uL
    • GFPT1-3 - 74.8 ng/uL
    • GFPT2-17 - 61.6 ng/uL
    • GFPT2-18 - 65.9 ng/uL
    • GFPT2-26 - 64.2 ng/uL
  • Ran a 1% agarose gel with 1-1, 1-2, 1-3, 2-17, 2-18, 2-26 plasmid miniprep samples and Hyperladder I
  • Prepared a 1:2 dilution of GFPT2 plasmid from 9/27 miniprep
  • Treated 5uL of diluted plasmid with 5uL of water, BL21 protein, topo protein, topo D168A protein
  • Incubated 30 minutes at 37C
  • Ran a 1% agarose gel with protein treated target plasmid samples and Hyperladder I
  • Digested GFPT2 plasmid with X (let run at 37C for 30 minutes)
  • Used DNA clean up kit on digested GFPT2
  • Nanodropped digested GFPT2:
    • GFPT2(X) - 39.85 ng/uL
  • Prepared DNA seq samples using VF2 and VR
  • Sample# - PrimerPair - DNA sample (sample 1, GFPT2 uncut + VF2; sample 2, GFPT2 uncut + VR)
    • 1/2 - FWD/REV - uncut GFPT2 plasmid
    • 3/4 - FWD/REV - cut GFPT2 plamid
    • 5/6 - FWD/REV - 2:1 uncut:cut GFPT2 plasmid mixture
    • 7/8 - FWD/REV - 1:1 uncut:cut GFPT2 plasmid mixture
    • 9/10 - FWD/REV - 1:2 uncut:cut GFPT2 plasmid mixture
    • 11/12 - FWD/REV - 1-2 miniprep sample from 9/19 double transformations
    • 13/14 - FWD/REV - 1-2I
    • 15/16 - FWD/REV - 1-3
    • 17/18 - FWD/REV - 1-3I
    • 19/20 - FWD/REV - 2-1
    • 21/22 - FWD/REV - 2-1I
    • 23/24 - FWD/REV - 2-2
    • 25/26 - FWD/REV - 2-2I
    • 27/28 - FWD/REV - 2-3
    • 29/30 - FWD/REV - 2-3I

October 1

  • Minipreps of:
    • J61011 + S + L + ALPHA 2B
    • J61100 + S + L + ALPHA 2B
    • J61100 + S + L + ALPHA 2C
    • PLUX2 + RBS + ALPHA 1A
    • PLUX2 + RBS + ALPHA 1A (2)
    • PLUX + S + L + ALPHA 2C
    • J61101 + S + L + ALPHA 1A
    • J61101 + S + L + ALPHA 1A (2)
    • PLUX2 + RBS + S + L + ALPHA 2B
    • PLUX2 + RBS + S + L + ALPHA 2B (2)
    • J61101 + S + L + ALPHA 2C
    • PLUX + S + L + ALPHA 2B
  • Ran Twice when finding concentrations:
    • J61101 + S + L + ALPHA 1A
    • J61101 + S + L + ALPHA 2B
  • Restricted all the above and:
    • Strep + Linker + OMEGA
    • Strep + Linker-4 + OMEGA
  • with:
    • X+P
  • Ligated into pSB1C3 shipping vector and transformed into BL21(DE3) cells.
  • Prepared all 14 above samples for sequencing.

October 2

  • Prepared PCR tubes with 10uL Topo1 4 (25ng/uL topo d168a in the shipping vector), 10uL GFPT2-26 (25ng/uL GFPT2 in the shipping vector), and 10uL GFPT1-3 (25ng/uL GFPT1 in the shipping vector)
  • Labelled the tubes K891234, K891999, K891000 respectively and shipped overnight to iGEM Headquarters
Retrieved from "http://2012.igem.org/Team:Arizona_State/Notebook"