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From 2012.igem.org

June 2012 S M T W T F S           1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

July 2012 S M T W T F S 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

August 2012 S M T W T F S       1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

September 2012 S M T W T F S             1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

October 2012 S M T W T F S   1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

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)

• 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
  • No picture

June 18

• Digest: Lac Z (X and P) and p + lacO (S and P)
• Protocol: http://openwetware.org/wiki/Haynes:Assembly101
• Gel extraction - Sigma Aldrich Gel Kit

June 19

• Ligation
  • P+lacO+Lac Z (samples still need to be tested)
  • Control (No DNA)
• 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

• PCR
  • Lac Z
    • Primers: Standard BBa F and R primers (http://partsregistry.org/Primers/Catalog)
    • 1000 fold dilution DNA used
    • Protocol
      • PCR water - 74.4 uL
      • 1000 fold dilution DNA - 6 uL
      • dNTP (10mM) - 2.4 uL
      • F and R primers (10 uM) - 6 uL each
      • 5x HF buffer - 24 uL
      • Phusion DNA polymerase - 1.2 uL
• Digest
  • P+Lac O
    • S and P
    • Treated with Antartic Phosphatase (avoids religation)
• 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: 2.554 ng/uL
  • Constitutive promoter 2: 2.345 ng/uL
  • T7 promoter 1: 3.369 ng/uL
  • T7 promoter 2: 3.049 ng/uL

June 22

• PCR clean up (Sigma Aldrich Kit) Lac Z samples
• Digest Lac Z with X and P
• Nano Drop the PCR clean samples:
  • Lac O: 2.554 ug/uL
  • Lac Z: 3.369 ug/uL
• Gel
  • 
• PCR
  • Lac Z (25ul rxn)
    • Primers: Standard BBa F and R primers (http://partsregistry.org/Primers/Catalog)
    • Varied Concentration DNA samples
    • Syzygy Mean Green MM
    • Lac Z samples - 10 fold D, 100 fold D, 1000 fold D and Regular DNA.

• PCR cleaned samples
• 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

• Third attempt of putting the bricks together. And samples were plated on X-gal plates to show that the construct worked.

• 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 12

• Searched for split beta gal fragment. Alpha fragment (BBa_I732006) was found on the distribution plates and transformed (NEB 10B).

July 14

• Designed primers to remove the stop codon on the alpha fragment. Also attained two flexible linkers from Dr. Haynes and transformed them.

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.

• Plate 1: pipet tip, colonies are in wells, neither distinct nor segregated
• Plate 2: inoculating loop, numerous non-distinct colonies on the plate

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

• Ran a miniprep of streptavidin BBa_K283010
• Protocol from Zymo Research's Zyppy Plasmid Miniprep Kit
• 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.
  

• 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.

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.

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

• Restricted PLFlex and PLFlex 4 with ecori and xbai.
• Restricted Bgala with Xbai and psti
• Used Hayne's Lab Protocol
• 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