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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)
• 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 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.
  

• 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

• 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

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.