IGEM:IMPERIAL/2008/New/Protocols/XL1-Blue preparation: Difference between revisions
m (New page: {{Imperial/StartPage}} ==Preparation of XL1-Blue Electrocompetent cells== ===Aims=== Preparation of ''E. coli'' cells for the cloning of Biobricks and construct construction ===Equipmen...) |
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==Preparation of XL1-Blue Electrocompetent cells== | ==Preparation of XL1-Blue Electrocompetent cells== | ||
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===Equipment=== | ===Equipment=== | ||
Centrifuge | 4,000RPM Centrifuge | ||
Sterile Centrifugation bottles | Sterile Centrifugation bottles | ||
50ml Tubes | |||
Large Flasks | Large Flasks | ||
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===Protocol=== | ===Protocol=== | ||
Keep Everything Cold where possible | Keep Everything Cold where possible - if using a carbon fibre rotor, you may want to put it in a cold room after innoculation. | ||
Set aside an afternoon for this, starting the culture in the morning | Set aside an afternoon for this, starting the culture in the morning | ||
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#Grow up a culture of ''E. coli'' XL1-blue cells overnight | #Grow up a culture of ''E. coli'' XL1-blue cells overnight | ||
#Add | #Add 40mL of overnight culture to 1 litre of LB medium (containing 20μg/ml Tetracycline) | ||
#Test OD immediately after innoculating the litre flask. | |||
#Grow cells while mixing at at least 225rpm until the culture reaches an OD<sub>600nm</sub> of 0.5-0.6 (1.6-1.9×10<sup>8</sup>cells/ml) | #Grow cells while mixing at at least 225rpm until the culture reaches an OD<sub>600nm</sub> of 0.5-0.6 (1.6-1.9×10<sup>8</sup>cells/ml) | ||
##First doubling may take 1 hour but doublings after that should be every 20-30 mins, so check often! | |||
##First doubling may take 1 hour but doublings after that should be | |||
#When OD is 0.5-0.6, transfer the culture to 2 sterile 500mL centrifugation bottles and cool on ice for a few minutes | #When OD is 0.5-0.6, transfer the culture to 2 sterile 500mL centrifugation bottles and cool on ice for a few minutes | ||
#Pellet cells in a centrifuge at | #Pellet cells in a centrifuge at 4,000g for 15 mins | ||
#Quickly but carefully pour off the supernatant then carefully resuspend the cells in 10mL of ice cold ddH<sub>2</sub>O | #Quickly but carefully pour off the supernatant then carefully resuspend the cells in 10mL of ice cold ddH<sub>2</sub>O | ||
#Fill both tubes to about 350mL with ice cold ddH<sub>2</sub>O | #Fill both tubes to about 350mL with ice cold ddH<sub>2</sub>O | ||
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##While repelleting, fill the dry ice bath and set up Eppendorf tubes (approximately 50) in a rack in the dry ice bath | ##While repelleting, fill the dry ice bath and set up Eppendorf tubes (approximately 50) in a rack in the dry ice bath | ||
#Pour off the supernatant and resuspend the cells in 20mL of 10% glycerol (resuspend one pellet then transfer the solution to the other bottle and resuspend the second pellet) | #Pour off the supernatant and resuspend the cells in 20mL of 10% glycerol (resuspend one pellet then transfer the solution to the other bottle and resuspend the second pellet) | ||
#Transfer the cells and glycerol solution to a sterile 50mL centrifuge tube and pellet for 15 mins at | #Transfer the cells and glycerol solution to a sterile 50mL centrifuge tube and pellet for 15 mins at 4,000g | ||
#Pour off supernatant and resuspend pellet in 2mL of 10% glycerol | #Pour off supernatant and resuspend pellet in 2mL of 10% glycerol | ||
#Pipette 50μL aliquots into the Eppendorfs in the dry ice bath | #Pipette 50μL aliquots into the Eppendorfs in the dry ice bath | ||
##Freeze on dry ice | ##Freeze on dry ice | ||
##Depending on pipetting accuracy, between 50 and 60 aliquots should be made | ##Depending on pipetting accuracy, between 50 and 60 aliquots should be made | ||
## | ##Using a repeating pipette makes this process much faster and reduces risk of contamination | ||
#Store at -70°C | #Store at -70°C | ||
{{Imperial/EndPage}} | {{Imperial/EndPage}} |
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