Team:Columbia-Cooper-NYC/Columbia notebook 2
From 2012.igem.org
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(→Monday, 20th) |
(→Tuesday, 21st) |
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*# High-copy Amp/CAM plasmid-PSB1AC3 (P1, 9A) | *# High-copy Amp/CAM plasmid-PSB1AC3 (P1, 9A) | ||
*# High-copy Amp/TET plasmid-PSB1AT3 (P1, 13A) | *# High-copy Amp/TET plasmid-PSB1AT3 (P1, 13A) | ||
+ | * 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 | ||
+ | **# old TET high copy plasmid | ||
+ | **# old Kill gene | ||
+ | **# Upps 5 | ||
+ | **# IPTG inducible promoter | ||
+ | ** Second row | ||
+ | **# 2 log ladder | ||
+ | **# newly digested TET high copy plasmid | ||
+ | **# newly digested Kill gene | ||
+ | **# Upps 1 | ||
+ | * Ran gel for 25 minutes at constant 150V | ||
+ | * Took picture under UV light |
Revision as of 16:00, 22 September 2012
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, 4th
- Streaked all 6 Uppsala (labelled below) parts from stabs onto plates:
- Upps 1: pSB1K3-B0034-YF1-B0034-FixJ
- Upps 2: pSB1K3-YF1
- Upps 3: pSB1K3-FixJ
- Upps 4: pSB1C3-PfixK2
- Upps 5: pSB1A3-amilCP
- Upps 6: pSB1C3 - amilGFP
- Prepared glycerol stock for phyB (ETHZ)
- Isolated phyB by following standard plasmid isolation protocol
- Electroporated fphA and ho1 from Germany
- Created LB/TET, LB/CAM, LB/Amp, LB/Kan plates
Sunday, 5th
- Pulled colonies from 6 iGEM parts and ho1 from Germany
- Streaked fphA from Germany
- Pulled colonies for Upps 2, 3, 4, 5, 6
- Streaked Upps 1, 3, 4, 6
- Parafilmed all plates and placed in 4C
- Sorted and threw out unnecessary plates
Monday, 6th
- Pulled colonies from fphA and Upps 1
- Isolated plasmids for the following samples following standard plasmid isolation protocol:
- 6 iGEM parts
- Upps 2-6
- ho1
- Reconfirmed that TET plates are valid
Tuesday, 7th
- Prepared glycerol stock and isolated the plasmid using standard plasmid isolation protocol for following:
- fphA from Germany
- Upps 1 (pSB1K3-B0034-YF1-B0034-FixJ)
- Prepared digestion by measuring OD at 260nm for following:
- Upps 1
- Upps 4
- Upps 5
- Upps 6
- Kill Gene
- Inducible promoter containing plasmid
- Prepared digestions and ligated with following setup using the 3A assembly protocol provided from Bioline:
- Upstream: Upps 4; Downstream: Upps 5; Destination Plasmid: High Copy TET plasmid
- Upstream: Upps 4; Downstream: Upps 6; Destination Plasmid: High Copy TET plasmid
- Upstream: Upps 4; Downstream: Kill gene; Destination Plasmid: Low Copy CAM plasmid
- Upstream: Inducible promoter; Downstream: Upps 5; Destination Plasmid: High Copy TET plasmid
- Upstream: Inducible promoter; Downstream: Upps 6; Destination Plasmid: High Copy TET plasmid
- Upstream: Inducible promoter; Downstream: Kill gene; Destination Plasmid: High Copy TET plasmid
Wednesday, 8th
- Pulled colony from GFP streak
- Isolated following using standard plasmid isolation protocol:
- Low Copy CAM plasmid
- High Copy TET plasmid
- Inducible promoter
- Checked optical density and applied necessary dilutions for GFP
- Prepared two samples of GFP: sample with IPTG and sample without IPTG
- Place samples in shaker to grow overnight
Thursday, 9th
- Diluted GFP solutions to match proper optical density
- Pulled colony for ho1 as backup and place in 37C inbucator
Friday, 10th
- Remade 200 mL each of antibiotic solutions for CAM, TET, Kan, Amp
- Conducted digestions and ligations using the 3A assembly method following the protocols provided by bioline of following:
- Upstream: Upps 4; Downstream: Kill gene; Destination Plasmid: High Copy TET plasmid
- Upstream: Inducible promoter; Downstream: Upps 5; Destination Plasmid: Low Copy CAM plasmid
- Upstream: Inducible promoter; Downstream: Upps 6; Destination Plasmid: Low Copy CAM plasmid
- Upstream: Inducible promoter; Downstream; Kill gene; Destination Plamid: Low Copy CAM plasmid
- Followed the butanol purification protocol for ligated material containing inducible promoter from 7th and 10th
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
Tuesday, 14th
Note: Work done below was conducted at the Kanbar Lab at the Cooper Union
- Prepared CAM from .250g of 25mg/mL CAM powder with 10mL of EtOH
- Prepared LB/glucose media
- Heat shocked DH5α competent cells with Inducible promoter/GFP/Low Copy CAM plasmid
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
Note: conducted the below procedures at the Kanbar Lab
- Observed no growth for inducible promoter/GFP/low copy CAM plasmid
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
Friday, 17th
Note: IPTG induced I-U5 and I-U6 appear to give no color change (no expression)
- Electroporate the following genes:
- Upps 4-Upps 6-High copy TET (sparked first time, redid trial)
- Upps 4-Kill-High copy TET
- Upps 4-Kill-Low copy CAM
- pUC19 control DNA (AMP resistance)
- Placed all samples in 37°C overnight
Note2: Only adding 25µl of competent cells instead of 50µl mentioned in the
Saturday, 18th
- Observed none of the electroporated DNA grew, but observed colonies for control
- Placed all samples back in the 37°C incubator
Monday, 20th
- Measured the optical density for the following:
- Upps 6 (with inducible promoter)
- Kill gene
- GFP
- Upps 5 (with inducible promoter)
- Diluted the concentrated primer into a primer stock that would be used for DNA sequencing
- Sent following DNA for sequencing (at Genewiz)
- Inducible promoter-GFP-Low Copy CAM plasmid
- Inducible promoter-Upps 5-Low Copy CAM plasmid
- Inducible promoter-Upps 6-Low Copy CAM plasmid
- Inducible promoter-Kill gene-Low Copy CAM plasmid
- Analyzed gel and concluded following:
- The digestion for TET high copy plasmid did not work
- The digestion for Upps 4, 5, 6 appeared to have worked
- All other digestions are inconclusive results
Tuesday, 21st
- Made additional Amp/CAM and Amp/TET plates
- Measured the optical density for the following:
- Upps 1
- Kill gene
- High copy TET plasmid
- Followed the standard digestion protocol provided by bioline or the following:
- Upps 1 (Upstream)
- Kill gene (Downstream)
- High copy TET plasmid (Destination plasmid)
- Re-organized iGEM boxes by throwing out previous digestions of High Copy TET plasmids
- Re-hydrated following biobricks from standard rehydration protocol:
- Constitutive promoter-BBa_J130002 (P1, 13B)
- Low-copy TET plasmid-PSB3T5 (P1, 7C)
- High-copy CAM plasmid-PSB1C3 (P1, 3A)
- High-copy Amp/CAM plasmid-PSB1AC3 (P1, 9A)
- High-copy Amp/TET plasmid-PSB1AT3 (P1, 13A)
- 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
- old TET high copy plasmid
- old Kill gene
- Upps 5
- IPTG inducible promoter
- Second row
- 2 log ladder
- newly digested TET high copy plasmid
- newly digested Kill gene
- Upps 1
- First row
- Ran gel for 25 minutes at constant 150V
- Took picture under UV light