Team:OU-Norman OK/Project/Notebook
From 2013.igem.org
02/12/13
stuff stuff stuff stuff
Back to top02/13/13
pSB1C3
Dilution 1:1000
58 Colonies
Back to top02/11/13
Transforming Competent Cells
We transformed TOP10 chemically competent cells using plasmids.
Resistance | Plasmid ID | Original Concentration (ng/μL) |
---|---|---|
Kanamycin | pSB1K3 | 62 |
Kanamycin | p34KM | 40 |
Chloramphenicol | pSB1C3 | 43 |
Ampicillin | pSB1A3 | 75 |
TOP10 competent cells in 100μL alliquots (x5) were thawed on ice and resuspended.
100ng of plasmid were added to cells
Cells were placed on ice for 20 minutes
Cells were transformed to a 42°C waterbath for 60 seconds
Plasmids were diluted to 20μL of 10μg/μL
p34KM
pSB1C3
pSB1A3
pSB1K3
Back to topBuffers for Preparing Competent E. coli
02/4/13
TFB1: pH: 5.8/ Sterile Filter
Chemicals | Concentration (mM) |
---|---|
RbCl | 100 |
MnCl2 | 50 |
Potassium Acetate | 30 |
CaCl2 | 10 |
Glycerol | 15% by Weight |
TFB2 ph:6.8 (Use KOH to adjust)/ Sterile Filter
Chemicals | Concentrations (mM) |
---|---|
MOPS | 10 |
RbCl | 10 |
CaCl2 | 75 |
Glycerol | 15% by Weight |
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Making Antibiotic Stocks
02/1/13
Antibiotics are used to isolate organisms into which plasmids containing antibiotic resistance genes have been transformed.
Make antibiotic plates with the following specs.
Antibiotic | Concentration (µg/mL) | Color Code |
---|---|---|
Ampicillin | 100 | Orange |
Chloramphenicol | 35 | Green |
Kanamycin | 50 | Red |
Tetracycline | 15 | Yellow |
Stocks should be made at 1000x concentration, so that making 1L of plate medium will require only 1mL of stock antibiotic solution.
Stocks of antibiotics are made at the following concentrations
Ampicillin | 100 mg/mL |
Chloramphenicol | 35 mg/mL |
Kanamycin | 30 mg/mL |
Tetracycline | 15 mg/mL |
50mL of stock were prepared and split into several 15mL tubes
Stocks should be stored in the refrigerator at 4°C
Back to top01/25/13
Restreaked TOP10 cells on LB plates for isolation of single colonies.
Back to top01/23/13
Sealed TOP10 E. coli cells on LB agar plate. Stored in 37°C incubator
Back to top01/18/13
Poured LB agar plates
Back to top01/16/13
Making Agar Plates
LB agar is used to grow our stocks of Escherichia coli
Mix Components in 1L of dH2O until dissolved. Spilt 1000mL solution into two flask. Cap flask with aluminum to prevent spilling of solution. Autoclave on slow exhaust for 20 minutes. Keep liquid in 65°C water bath to prevent setting until 18-Jan.
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