Team:Imperial College/XL1 Blue
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(New page: {{Imperial/StartPage2}} ==Preparation of XL1-Blue Electrocompetent cells== ===Aims=== Preparation of ''E. coli'' cells for the cloning of Biobricks and construct construction ===Equipme...) |
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{{Imperial/StartPage2}} | {{Imperial/StartPage2}} | ||
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+ | {{Imperial/Box1|Preparation of XL1-Blue Electrocompetent cells| | ||
+ | ======Aims====== | ||
Preparation of ''E. coli'' cells for the cloning of Biobricks and construct construction | Preparation of ''E. coli'' cells for the cloning of Biobricks and construct construction | ||
- | + | ======Equipment====== | |
- | ===Equipment=== | + | |
- | + | ||
4,000RPM Centrifuge | 4,000RPM Centrifuge | ||
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Stripettes | Stripettes | ||
- | ===Reagents=== | + | ======Reagents====== |
- | + | ||
1 litre of LB medium | 1 litre of LB medium | ||
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Dry ice bath | Dry ice bath | ||
- | ===Protocol=== | + | ======Protocol====== |
- | + | ''Keep everything cold where possible - if using a carbon fibre rotor, you may want to put it in a cold room after innoculation.'' | |
- | Keep | + | |
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 40mL of overnight culture to 1 litre of LB medium (containing 20μg/ml Tetracycline) | #Add 40mL of overnight culture to 1 litre of LB medium (containing 20μg/ml Tetracycline) | ||
- | #Test OD immediately after | + | #Test OD immediately after inoculating 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! |
#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 4,000g for 15 mins | #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 | ||
- | # | + | #*Make sure the pellet is fully resuspended! |
#Repellet the cells (as before) and again discard the supernatant | #Repellet the cells (as before) and again discard the supernatant | ||
#Resuspend cells again in 10mL of ddH<sub>2</sub>O, then fill both tubes up to about 150mL with ice cold ddH<sub>2</sub>O | #Resuspend cells again in 10mL of ddH<sub>2</sub>O, then fill both tubes up to about 150mL with ice cold ddH<sub>2</sub>O | ||
#Repellet the cells (as before) | #Repellet the cells (as before) | ||
- | # | + | #*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 4,000g | #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 |
- | # | + | #*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|Protocols|Protocols}} |
Latest revision as of 17:40, 28 October 2008
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