Team:Arizona State/Notebook

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Revision as of 03:54, 3 October 2012

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Note from Dr. Haynes: Rohit, please transfer all of the Notebook entries from the OWW Wiki
http://openwetware.org/wiki/Haynes_Lab:Notebook/ASU_iGEM
To this page. Follow the format below. I added a couple of sections to help you get started.

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)

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

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.
- Insert picture here.

August 13

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.

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

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.

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.

@@ Line 183: / Line 183: @@
 * 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.
+* Transformed Magainin insert into digested pUC 19 plasmid. Failed. Probably too much X-gal on plate. <Br\>
-** Insert picture here
+[[File: ASUiGEM2012_gel081312.jpeg|200px]]
 ==August 15==