Team:Wash U/Protocol
From 2009.igem.org
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# Begin by thawing all DNA along with NEBuffer 2 and BSA. | # Begin by thawing all DNA along with NEBuffer 2 and BSA. | ||
# Label three separate PCR tubes upstream, downstream, and destination plasmid. To each tube 500ng of respective DNA and dilute to 42.5uL. Add 5uL of NEBuffer 2 and 0.5uL of BSA to each tube. | # Label three separate PCR tubes upstream, downstream, and destination plasmid. To each tube 500ng of respective DNA and dilute to 42.5uL. Add 5uL of NEBuffer 2 and 0.5uL of BSA to each tube. | ||
- | # Add 1uL of the first appropriate enzyme to each tube (see [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.jpg chart] for correct enzyme. Then add 1uL of second appropriate enzyme to each tube. | + | # Add 1uL of the first appropriate enzyme to each tube (see [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.jpg chart] for correct enzyme). Then add 1uL of second appropriate enzyme to each tube. |
# Flick the tubes to mix contents and microcentrifuge for a few seconds to collect liquid in bottom of tube. | # Flick the tubes to mix contents and microcentrifuge for a few seconds to collect liquid in bottom of tube. | ||
# Incubate the three digests at 80C for 20 minutes to deactivate the restriction enzymes. The digestion portion of the procedure is now completed. You may wish to store the products at -20C or proceed directly to the ligation step. | # Incubate the three digests at 80C for 20 minutes to deactivate the restriction enzymes. The digestion portion of the procedure is now completed. You may wish to store the products at -20C or proceed directly to the ligation step. |
Revision as of 15:47, 10 July 2009