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| - | == '''Electroporation Protocol''' | + | <html><div z-index:100><img src="https://static.igem.org/mediawiki/2014/3/31/Electroporation_Picture_2.PNG" width="350" height="200" align="right"></div></html> |
| - | ==
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| - | '''Purpose
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| - | '''Electroporation promises better transformation efficiency than starvation protocols and takes less time and is easier to execute. This protocol outlines how to use the Life Technologies Cell-Porator machine found in Bindley 222.
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| - | '''Solutions
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| - | '''Growth medium: LB medium containing 0.5 M sorbitol
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| - | Washing solution: 0.5 M sorbitol, 0.5 M mannitol, 10 % glycerol
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| - | Electroporation solution: 0.5 M sorbitol, 0.5 M mannitol, 10 % glycerol
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| - | Outgrowth medium: LB medium containing 0.5 M sorbitol and 0.38 M mannitol
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| - |
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| - | '''Materials
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| - | '''• Life Technologies Cell-Porator
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| - | • Cell-Porator chambers (4)
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| - | • Competent cells
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| - | • Micropipette and tips
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| - | • SOC solution
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| - |
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| - | '''Methods
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| - | '''
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| - | Making electrocompetent cells:
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| - | 1. Dilute an overnight culture of Bacillus subtilis 16-fold in growth medium and grow at 37 °C to an O.D.600 of 0.85-0.95.
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| - | 2. Cool the cells on ice-water for 10 min. and harvest by centrifugation at 4 °C and 5000 x g for 5 min.
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| - | 3. Wash cells four times in ice-cold electroporation medium.
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| - | 4. Suspend the cells in 1/40 of the culture volume of the electroporation solution with a cell concentration of
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| - | 1-1.3 x 1010 cfu/ml.
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| - | 5. The competent cells can be stored at –80 °C until use with some decrease in transformation efficiency.
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| - |
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| - | Electroporation of cells:
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| - | 1. Add 1 µl (50 ng/µl) plasmid DNA to 60 µl of electrocompetent cells. Homogenize by gently mixing with pipette several times. Transfer mixture into a prechilled cuvette. Incubate for 1-1.5 min.
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| - | 2. Place ice water in chamber under cell holder rack
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| - | 3. Place electroporator chambers on ice
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| - | 4. Turn on voltage booster and controller using switches on back
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| - | 5. Set to these conditions:
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| - | 6. Voltage Booster: Level 4
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| - | 7. Charge rate: fast
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| - | 8. Volts: Low
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| - | 9. Capacitance: 330
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| - | 10. Pipette 20μL of competent cells with DNA between electrodes
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| - | 11. Fill all 4 slots in chamber
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| - | 12. Close chamber, plug in power source
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| - | 13. Make sure chamber selector is on the correct chamber
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| - | 14. Press up on the controller until voltage reaches 405
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| - | 15. At 405, switch from charge to arm and press trigger for 1 second
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| - | 16. Release trigger and switch arm to charge *If there is too much salt in the DNA, a pop will happen and the liquid will not be suspended between the electrodes
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| - | 17. Immediately add 1 ml outgrowth medium and incubate for 3 h at 37 °C.
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| - | 18. Plate onto selective LB agar plates and incubate overnight at 37 °C.
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| - | <html><div z-index:100><img src="https://static.igem.org/mediawiki/2014/9/91/SAM_0252.jpg" width="350" height="200" align="right"></div></html>
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| - | == '''Phytosiderrophore Production Assay
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| - | ''' ==
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| - |
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| - | '''O-CAS Siderophore Assay
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| - | '''
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| - |
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| - | '''Introduction'''
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| - | The CAS assay includes inhibitory compounds that negatively impact the growth of gram-positive bacteria (e.g. Bacillus subtilis) and fungi. Therefore, the O-CAS assay must be used.
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| - |
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| - | '''Materials
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| - | '''
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| - | • 20% glycerol solution
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| - | • Chrome azurol S (CAS)
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| - | • HDTMA
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| - | • PIPES
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| - | • Agarose
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| - |
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| - | '''Procedure
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| - | '''
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| - | 1. Plates containing growth medium were stored at 28°C for 24h
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| - | 2. Bacteria were preserved in 20% glycerol solution at -20°C
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| - | 3. The CAS medium is prepared as follows:
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| - | a. Chrome azurol S (CAS) 60.5 mg
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| - | b. hexadecyltrimetyl ammonium bromide (HDTMA) 72.9 mg
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| - | c. Piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) 30.24 g
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| - | d. 1 mM FeCl3• 6H2O in 10 mM HCl 10 mL.
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| - | e. Agarose (0.9%, w/v) was used as gelling agent
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| - | 4. Apply 10mL of medium over standard Petri dish (or 30mL over large Petri dish)
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| - | 5. Color will change after 15 min if siderophores are detected
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