Team:Arizona State/Notebook

From 2012.igem.org

(Difference between revisions)
(→August 15)
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Tried gel for all gel-isolated fragments. Failed. Did not get a band in the 2000 bp region. Only got things below 200 bp.<BR\>
Tried gel for all gel-isolated fragments. Failed. Did not get a band in the 2000 bp region. Only got things below 200 bp.<BR\>
[[Image:ASUiGEM2012_gel081612.jpeg|200px]]
[[Image:ASUiGEM2012_gel081612.jpeg|200px]]
 +
 +
==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==
==October 1==

Revision as of 04:31, 3 October 2012


June 2012
S M T W T F S
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July 2012
S M T W T F S
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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
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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
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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
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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.

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

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.
Retrieved from "http://2012.igem.org/Team:Arizona_State/Notebook"