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Team:HKU HongKong/Data/Protocols.html
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Molecular Cloning Protocols</font></span></h2> | Molecular Cloning Protocols</font></span></h2> | ||
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Revision as of 14:45, 23 September 2012
Team:HKU HK
From 2011.igem.org
Molecular Cloning Protocols
Transforming Competent E.coli with the
Plasmid:
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Take out the competent E-coli cells from the -80 freezer. (Keep all tubes on ice).
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Add 1uL of the plasmid DNA in 100uL competent cells (if from Kit). For transformation of ligated product, add 10uL of the ligated plasmid DNA in 100uL competent
cells.
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Incubate on ice for 10 min.
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Place in water bath at 42°C for 90s.
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Place immediately back on ice for at least 2 min.
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Add 800uL LB broth. Incubate for 1hr at 37°C shaker.
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Centrifuge at 130rpm for 5min to remove supernatant. Re-suspend the pellet in about 10uL of supernatant.
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Spread the entire re-suspended pellet on ampicillin agar dishes.
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Incubate for 12-16 hours at 37 °C.
Notes:
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Colonies grown on plate were selected for colony PCR screening.
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Correct colonies were then cultured in 5 mL LB Broth with 0.5uL ampicillin for subsequent screening or mass culturing.
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Mass culturing involved adding 200uL of the culture into 35mL LB Broth with 35uL ampicillin.
Miniprep to Extract the Plasmid from E.coli (QIAprep Spin Miniprep Kit):
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Transfer some of the 5mL bacterial culture into a microcentrifuge tube. Pellet by centrifugation at 13,500 rpm for 1 min. Repeat till all the culture has been pelleted.
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Resuspend the pellet in 250uL Buffer P1.
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Add 250uL Buffer P2 (Lysis Buffer). Mix thoroughly by inverting the tube 4-6 times. Do not allow prolonged lysis.
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Immediately add 350uL Buffer N3 (Neutralization Buffer). Mix immediately by inverting the tube.
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Centrifuge for 10 minutes at 13,500 rpm.
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Apply the resulting supernatant to the QIAprep spin column by pipetting. Centrifuge for 1 minute at 13,500 rpm.
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Wash the QIAprep spin column by applying 0.75mL Buffer PE. Centrifuge for 1 minute at 13,500 rpm.
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Centrifuge for an additional 1 minute to remove residual ethanol.
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Place the QIAprep spin column in microcentrifuge tube. Elute DNA by adding 50uL warm H2O.
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Centrifuge for 1 minute at 13,500 rpm.
