Team:Columbia-Cooper-NYC/Columbia notebook 2

From 2012.igem.org

(Difference between revisions)
(Monday, 13th)
(Week 7)
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''Note 3: Original DNA for Upps 4-Upps 5-TET was pink''
''Note 3: Original DNA for Upps 4-Upps 5-TET was pink''
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* Placed transformed samples in growth media and placed in 37°C shaker
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* Made 60ml of 1% agar gel for running gel electrophoresis (2 rows of 12 wells each noted below for gel) to check digestion and ligation
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** First row
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**# 2 log ladder
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**# Upps 4
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**# blank
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**# High Copy TET plasmid
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**# Upps 4 (2)
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**# Upps 6
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**# High Copy TET plasmid (2)
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**# Upps 4 (3)
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**# Kill gene
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**# High Copy TET plasmid (3)
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**# PCB
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**# High Copy TET plasmid (4)
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** Second row
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**# 2 log ladder
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**# Inducible promoter-IPTG
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**# Kill gene (2)
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**# High Copy TET plasmid (5)
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**# Inducible promoter-IPTG (2)
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**# Upps 6 (2)
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**# High Copy TET plasmid (6)
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**# Inducible promoter-IPTG (3)
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**# Upps 5 (2)
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**# High Copy TET plasmid (7)
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* Ran gel for 25 minutes at constant 150V
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* Took picture under UV light
==== Wednesday, 15th ====
==== Wednesday, 15th ====
* Diluted bacteria cultures with following plasmids to 200x LB/CAM and placed in 37°C shaker
* Diluted bacteria cultures with following plasmids to 200x LB/CAM and placed in 37°C shaker

Revision as of 22:30, 16 August 2012

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Columbia Genetics Lab Notebook

July, 2012

Week 1

Thursday, 5th

  • Re-hydrated plasmids with 50µl of LB and Kanamycin solution
  • Stored solution at 37°C incubator overnight

Friday, 6th

  • Purified pET26b vector using standard DNA purification protocol

Week 2

Monday, 9th

  • Received kill gene Bba-K124017 from plate 3, 20M
  • Re-hydrated DNA according to standard iGEM re-hydration protocol

Tuesday, 10th

  • Contacted professors at Germany in hopes to receive copies of fungal phytochrome FphA

Wednesday, 11th

  • Received confirmation by professors at Germany for FphA to be sent to Columbia University
  • Conducted transformation using electroporation with competent bacteria (marked by resistance to Kanamycin)
    1. Control: 1µl of deionized water with abt. and 60µl of bacteria cells
    2. Variable: 1µl of re-hydrated kill gene and 60µl of bacteria cells
  • Placed both samples after electroporation into 200µl of preprepared LB
  • Placed samples in shaker 37°C for 30 minutes

Thursday, 12th

  • Grew 1 colony of transformed bacteria in 5mL of Kanamycin and LB solution
    • Note: Using pET20b vector over pET26b vector from glycerol stock solution

Friday, 13th

  • Isolated 4 samples of plasmid using standard plasmid isolation protocol
    1. 2 samples: kill gene
    2. 2 samples: pET20b vector

Week 3

Monday, 16th

  • Re-hydrated two biobrick parts in plasmid pSB2K3 according to standard iGEM re-hydration protocol
    1. BBa-I16009 (PcyA) from plate 1, 20F
    2. BBa-I16008 (ho1) from plate 2, 13J
  • Electroporated 1µl of each biobrick into separate E. coli at 1800V
  • Added 100µl LB broth into each sample
  • Placed samples at 33.4°C for 20 minutes
  • Samples were plated to be grown overnight

Tuesday, 17th

  • Placed 5ml each of LB/Kan into two centrifuge tube for PCB creation
    1. Label P: PcyA
    2. Label h: ho1
  • Placed samples in 37°C incubator

Wednesday, 18th

  • Purified ho1 and PcyA plasmids using standard DNA purification protocol
  • Placed purified DNA into glycerol stock (LB/Kan) and stored at -80°C

Thursday, 19th

  • Purified GFP using standard DNA purification protocol
  • Prepared glycerol stock solution (500µl GFP/500µl 80% glycerol) and stored at -80°C

Week 4

Tuesday, 24th

  • Re-hydrated four biobrick parts according to standard iGEM re-hydration protocol
    1. Inducible plasmid (pSB1AK3-J04500) from plate 4, 12A
    2. GFP (pSB1A2-E0040) from plate 1, 14K
    3. High copy plasmid pSB1T3-J044500 from plate 1, 7A
    4. Low copy plasmid (pSB3C5-J044500) from plate 1, 3C
  • Electroporated competent E. coli with each of the four above genes separetely

Wednesday, 25th

Thursday, 26th

Friday, 27th

Saturday, 28th

Week 5

August, 2012

Week 5

Week 6

Saturday, 11th

  • Reviewed the solutions for diluted GFP and observed no significant results
  • Reorganized samples in fridges and incubators

Week 7

Monday, 13th

  • Chemically transformed competent cells (BL21) with plasmids below using bioline protocol (used 1/2 of recommended amount)
    1. IPTG-Upps 5-Low Copy (CAM)
    2. IPTG-Upps 6-Low Copy (CAM)
    3. IPTG-Kill gene-Low Copy (CAM)
    4. CAM control plasmid
    5. PUC19 control plasmid
  • Electroporated competent cells (α-select) with plasmids below using bioline protocol
    1. Upps 4-Kill gene-High Copy (TET)
    2. Upps 4-Upps 5-High Copy (TET)
    3. Upps 4-Upps 6-High Copy (TET)
    4. PUC19 control plasmid
    5. High copy TET control plasmid

Note 1: TET control sparked

Note 2: Upps 4-Upps 5-TET sparked

Note 3: Original DNA for Upps 4-Upps 5-TET was pink

  • Placed transformed samples in growth media and placed in 37°C shaker
  • Made 60ml of 1% agar gel for running gel electrophoresis (2 rows of 12 wells each noted below for gel) to check digestion and ligation
    • First row
      1. 2 log ladder
      2. Upps 4
      3. blank
      4. High Copy TET plasmid
      5. Upps 4 (2)
      6. Upps 6
      7. High Copy TET plasmid (2)
      8. Upps 4 (3)
      9. Kill gene
      10. High Copy TET plasmid (3)
      11. PCB
      12. High Copy TET plasmid (4)
    • Second row
      1. 2 log ladder
      2. Inducible promoter-IPTG
      3. Kill gene (2)
      4. High Copy TET plasmid (5)
      5. Inducible promoter-IPTG (2)
      6. Upps 6 (2)
      7. High Copy TET plasmid (6)
      8. Inducible promoter-IPTG (3)
      9. Upps 5 (2)
      10. High Copy TET plasmid (7)
  • Ran gel for 25 minutes at constant 150V
  • Took picture under UV light

Wednesday, 15th

  • Diluted bacteria cultures with following plasmids to 200x LB/CAM and placed in 37°C shaker
    1. IPTG-Upps 5-Low Copy (CAM)
    2. IPTG-Upps 6-Low Copy (CAM)
    3. IPTG-Kill gene-Low Copy (CAM)
  • Purified above plasmids and CAM control plasmid using standard purification protocol and prepared glycerol stocks
  • Added appropriate buffers to IPTG-Upps 5 and IPTG-Upps 6 and centrifuged for 10 minutes to determine for a pellet
  • Measured OD 600 of following samples
    1. IPTG-Upps 5-Low Copy (CAM): .160A
    2. IPTG-Upps 6-Low Copy (CAM): .038A
  • Inserted 1µl of 1M IPTG into cultures
  • Placed all samples in 37°C overnight

Thursday, 16th

  • Measured OD 600 for 200x diluted bacterial solution containing plasmids with promoter inducible with IPTG
    1. IPTG-Upps 5-Low Copy (CAM): .040A
    2. IPTG-Upps 6-Low Copy (CAM): .032A
    3. IPTG-Kill gene-Low Copy (CAM): .028A
  • Noted that cell concentration was dense, decided to dilute solution with 75µl cells and 925µl LB/CAM solution
  • Placed diluted cell solution into 37°C incubator for 40 minutes
  • Inserted 1µl of 1M IPTG into cultures
  • Placed all samples in 37°C overnight