Team:Columbia-Cooper-NYC/Columbia notebook 2
From 2012.igem.org
Columbia Genetics Lab Notebook
July, 2012
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
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)
- Control: 1µl of deionized water with abt. and 60µl of bacteria cells
- 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
- 2 samples: kill gene
- 2 samples: pET20b vector
Monday, 16th
- Re-hydrated two biobrick parts in plasmid pSB2K3 according to standard iGEM re-hydration protocol
- BBa-I16009 (PcyA) from plate 1, 20F
- 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
- Label P: PcyA
- 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
Tuesday, 24th
- Re-hydrated four biobrick parts according to standard iGEM re-hydration protocol
- Inducible plasmid (pSB1AK3-J04500) from plate 4, 12A
- GFP (pSB1A2-E0040) from plate 1, 14K
- High copy plasmid pSB1T3-J044500 from plate 1, 7A
- Low copy plasmid (pSB3C5-J044500) from plate 1, 3C
- Electroporated competent E. coli with each of the four above genes separetely
- Created Kan, Amp, Cam, Tetra, and Amp/Kan plates
Wednesday, 25th
- Streaked pSB1T3-J04450
- Created LB solution with Kan or Amp or Cam
Thursday, 26th
- Prepared Glycerol stock for inducible promoter, GFP, and low-copy plasmid
- Picked a single colony from pSB1T3-J04450 and let it grow overnight in 37C
- Recorded and measured the DNA concentrations of following at 260nm:
- Kill gene
- PcyA
- ho1
- GFP
- Inducible promoter
- Low copy CAM plasmid
- Followed digestion and ligation protocol; setup explained below:
- Upstream: ho1; Downstream: PcyA; Destination plasmid: Low-copy CAM
- Upstream: Inducible promoter; Downstream: GFP; Destination plasmid: Low-copy CAM
- Upstream: Inducible promoter; Downstream: Kill; Destination plasmid: High-copy TET (pSB1T3)
- After completion, store samples at -20C
Friday, 27th
- Electroporated ligated samples from previous day: GFP, PCB, Kill
- Followed standard plasmid isolation protocol for pSB1T3-J04450 (TET plasmid)
Saturday, 28th
- Check plates from electroporation from previous day
Monday, 30th
- Stored pif3 and phyB that arrived from Sweden
- Relocated and reorganized the iGEM biobrick kit and glycerol stocks
- Picked colonies for following DNA:
- Inducible promotor (pSB1AK3_J04500)
- GFP (pSB1A2_E0040)
- Low copy CAM plasmid (pSB3C5_J04150)
- Added 10µl of ho1 and PcyA to 5ml of antibiotics
- Electroporated following DNA:
- Inducible promoter IPTG/kill gene in BL21 cells
- Inducible promoter IPTG/GFP in BL21 cells
- PCB in α cells
- Transferred successful electroporated cells to eppendorf tube with 100μL of LB and placed in 37C shaker
Tuesday, 31st
- Prepared glycerol stock of the following:
- GFP
- Inducible promoter (IPTG)
- Low copy CAM plasmid
- pcyA
- ho1
- Isolated ho1 and PcyA using standard plasmid isolation protocol
August, 2012
Wednesday, 1st
- Applied IPTG to samples of bacteria with GFP or kill gene and placed back in incubator at 37C
- Electroporated the following plasmids:
- IPTG inducible promoter/kill gene into BL21 cells
- IPTG inducible promoter/GFP into BL21 cells
- phyB into α select cells
- pif3 into α select cells
- Followed the PCR purification protocol for samples 1, 2 listed above
- Chemically transformed the following using standard heat shock protocol provided by Bioline:
- IPTG inducible promoter/kill gene into BL21 cells
- IPTG inducible promoter/GFP into BL21 cells
- Plated PCB onto LB/CAM plate
- Plated Pif3 and phyB on KAN plates
- Plated 500μL of GFP containing cells among two plates each LB/CAM (with and without IPTG)
- Placed all samples in 37C incubator
Thursday 2nd
- Selected colonies from PhyB from the LB/Kan plate
- Prepared CAM plates
- Selected colonies from GFP control
- Received agar stabs from Uppsala
Friday 3rd
- Placed 60μL of IPTG to half of control plate for reconfirmation of results
- Streaked GFP (with IPTG inducible promoter) and Pif3 on plates
Saturday, 11th
- Reviewed the solutions for diluted GFP and observed no significant results
- Reorganized samples in fridges and incubators
Monday, 13th
- Chemically transformed competent cells (BL21) with plasmids below using bioline protocol (used 1/2 of recommended amount)
- IPTG-Upps 5-Low Copy (CAM)
- IPTG-Upps 6-Low Copy (CAM)
- IPTG-Kill gene-Low Copy (CAM)
- CAM control plasmid
- PUC19 control plasmid
- Electroporated competent cells (α-select) with plasmids below using bioline protocol
- Upps 4-Kill gene-High Copy (TET)
- Upps 4-Upps 5-High Copy (TET)
- Upps 4-Upps 6-High Copy (TET)
- PUC19 control plasmid
- 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
- 2 log ladder
- Upps 4
- blank
- High Copy TET plasmid
- Upps 4 (2)
- Upps 6
- High Copy TET plasmid (2)
- Upps 4 (3)
- Kill gene
- High Copy TET plasmid (3)
- PCB
- High Copy TET plasmid (4)
- Second row
- 2 log ladder
- Inducible promoter-IPTG
- Kill gene (2)
- High Copy TET plasmid (5)
- Inducible promoter-IPTG (2)
- Upps 6 (2)
- High Copy TET plasmid (6)
- Inducible promoter-IPTG (3)
- Upps 5 (2)
- High Copy TET plasmid (7)
- First row
- Ran gel for 25 minutes at constant 150V
- Took picture under UV light
- Created TET and CAM plates
Wednesday, 15th
- Diluted bacteria cultures with following plasmids to 200x LB/CAM and placed in 37°C shaker
- IPTG-Upps 5-Low Copy (CAM)
- IPTG-Upps 6-Low Copy (CAM)
- 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
- IPTG-Upps 5-Low Copy (CAM): .160A
- 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
- IPTG-Upps 5-Low Copy (CAM): .040A
- IPTG-Upps 6-Low Copy (CAM): .032A
- 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