IGEM:PennState/Labbook/LucienWeiss: Difference between revisions
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<h2>'''May 15, 2007: General Procedures'''</h2> | <h2>'''May 15, 2007: General Procedures'''</h2> | ||
Miniprep | |||
#Spin down 3mL of saturated cell culture, remove supernatant | |||
#Resuspend pelleted bacteria in 250uL Buffer P1 and transfer to a 1.5mL micro centrifuge tube. | |||
#Add 250uL Buffer P2 and invert the tube gently 4-6 times to mix. | |||
#Add 350uL Buffer N3 and invert the tube immediately but gently 4-6 times. | |||
#*the solution should be come cloudy | |||
#Centrifuge for 10 minutes at 13,000 rpm in a table-top microcentrifuge | |||
#*allign tubes such that the cap clip is facing outward | |||
#decant supernatant into spin column | |||
#Centrifuge for 60s. Discard flow-through. | |||
#Add .75mL Buffer PE and centifuge for 60s. | |||
#Discard flowthrough and centrifuge for 60s | |||
#Place the spin column into a new 1.5mL microcentrifuge tube. | |||
#Add 30-50uL of water (8.5>pH>7) to the center of each spin column. | |||
#Let tubes stand vertically for 1min, then centrifuge for 1min. | |||
10uL Digest | 10uL Digest | ||
#2.5uL H2O | #2.5uL H2O |
Revision as of 10:34, 15 May 2007
Lucien Weiss Lab Notebook
- Penn State iGEM 2005-2007
- Penn State Undergraduate
- Email luw134@psu.edu
- Phone (814) 880 5909
March 12, 2007: Motility Assay
Experiment Aims
Test motility on a variety of surface hardnesses in order to determine optimal level to use for assays.
Procedure
Eiken Agar Plates were made using a modified version of general procedure 1. Increased agar concentrations were made to increase hardness of gel and reduce the speed of motility.
.20% Eiken Agar Plates:
- .25g Bactotryptone
- .20g NaCl
- .15g sodium citrate
- .05g Eiken Agar (for 20EikenAgar)
.30% Eiken Agar Plates:
- .25g Bactotryptone
- .20g NaCl
- .15g sodium citrate
- .075g Eiken Agar (for 30EikenAgar)
.40% Eiken Agar Plates:
- .25g Bactotryptone
- .20g NaCl
- .15g sodium citrate
- .10g Eiken Agar (for 40EikenAgar
- Constructs were transformed into RP3087
- YFP Sender Cells-Sender (LVA+)-RP3087 w/ [I14032+B0030+C0161 A3]+E0430
- Sender Cells-Sender-RP3087 w/ I14032+B0030+C0161 (B2)
- Receiver-RFP+Receiver-RP3087 w/ I13321+[J09855+B0033+J09274]
- Grown overnight at 37 degrees C on 50 micromolar Kan LB plates
- 3mL cultures inoculated off of the plate and grown at 30 degrees C until OD590~.45
- 3uL placed of receiver cells placed in various distances from 3uL inoculated sender colony.
Results:
.20% Eiken Agar Sender and Receivers | |
.30% Eiken Agar Sender and Receivers | |
.30% Eiken Agar with Degradation Tagged AHL | |
.40% Eiken Agar Sender and Receivers | |
Observations:
.20% agar plate difficult to handle as agar was extremely fragile. Results were diminished by heavy diffusion. .30% agar results in fair motility and is much easier to handle .40% agar results in no motility
Conclusions:
Future Experiments:
Retry .20% Agar experiments with set distances away from sender Experiment with temperature changes
March 13, 2007: Motility Assay
Experiment Aims
Test induced motility at a variety of distances from sender cells.
Procedure
Eiken Agar Plates were made using a modified version of general procedure 1. Agar concentrations were kept at .20%
.20% Eiken Agar Plates:
- .25g Bactotryptone
- .20g NaCl
- .15g sodium citrate
- .05g Eiken Agar (for 20EikenAgar)
- Constructs were transformed into RP3087
- YFP Sender Cells-Sender (LVA+)-RP3087 w/ [I14032+B0030+C0161 A3]+E0430
- Sender Cells-Sender-RP3087 w/ I14032+B0030+C0161 (B2)
- Receiver-RFP+Receiver-RP3087 w/ I13321+[J09855+B0033+J09274]
- Grown overnight at 37 degrees C on 50 micromolar Kan LB plates
- 3mL cultures inoculated off of the plate and grown at 30 degrees C until OD590~.45
- 3uL placed of receiver cells placed in various distances from 3uL inoculated sender colony.
Results:
.20% Eiken Agar Sender and Receivers | |
Observations:
Difficult to discern motility on .20% Eiken Agar plates
Conclusions:
Future Experiments:
Increase Agar Concentration
Experiment with temperature changes
March 16, 2007: Motility Assay
March 29-April 1, 2007: Institute of Biological Engineering
Poster: Bacterial Relay Race |
April 22, 2007: Project Proposal Presentations
<table|left>
Project Proposal: Pressure Sensing in Bacteria
<table|left>
Project Proposal: Bi and Sept Stable Oscillator
April 28, 2007: Synthetic Biology 3.0
May 15, 2007: General Procedures
Miniprep
- Spin down 3mL of saturated cell culture, remove supernatant
- Resuspend pelleted bacteria in 250uL Buffer P1 and transfer to a 1.5mL micro centrifuge tube.
- Add 250uL Buffer P2 and invert the tube gently 4-6 times to mix.
- Add 350uL Buffer N3 and invert the tube immediately but gently 4-6 times.
- the solution should be come cloudy
- Centrifuge for 10 minutes at 13,000 rpm in a table-top microcentrifuge
- allign tubes such that the cap clip is facing outward
- decant supernatant into spin column
- Centrifuge for 60s. Discard flow-through.
- Add .75mL Buffer PE and centifuge for 60s.
- Discard flowthrough and centrifuge for 60s
- Place the spin column into a new 1.5mL microcentrifuge tube.
- Add 30-50uL of water (8.5>pH>7) to the center of each spin column.
- Let tubes stand vertically for 1min, then centrifuge for 1min.
10uL Digest
- 2.5uL H2O
- 4.0uL Vector
- 0.5uL Insert
- 1.0uL Buffer X
- 1.0uL BSA
- 0.5uL Enzyme 1
- 0.5uL Enzyme 2
20uL Digest
- 2.0uL H2O
- 11.uL Vector
- 2.0uL Insert
- 2.0uL Buffer X
- 2.0uL BSA
- 0.5uL Enzyme 1
- 0.5uL Enzyme 2