Team:Wash U/Protocol
From 2009.igem.org
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=='''BioBrick Assembly'''== | =='''BioBrick Assembly'''== | ||
<font size="2"> | <font size="2"> | ||
- | :BioBrick Assembly is a standard protocol for combining two BioBrick parts and positioning them in a destination plasmid. | + | :BioBrick Assembly is a standard protocol for combining two BioBrick parts and positioning them in a destination plasmid. Specific cut sites for standard restriction enzymes are utilized to simplify the protocol and include EcoR1-HF, Xba1, Spe1 and Pst1. The assembly consists of two major parts: digestion and ligation. Note that BioBrick assembly requires purified, isolated DNA for all parts involved (upstream, downstream and destination plasmid). To view the full Biobrick Assembly manual, please click [http://ginkgobioworks.com/support/ here]. |
'''Materials''' | '''Materials''' | ||
* NEBuffer 2 | * NEBuffer 2 | ||
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'''Procedures''' | '''Procedures''' | ||
# 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. | + | # 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 | + | # 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. | ||
+ | # 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. | ||
+ | # Begin ligation by thawing 10X T4 DNA Ligase Reaction Buffer and enzyme. Agitate the buffer until all precipitate goes into solution. | ||
+ | # Add 11uL of H20 to a 200uL PCr tube. | ||
+ | # Add 2uL of each digest product to the new PCR tube. | ||
+ | # Add 2uL of 10X T4 DNA Ligase Reaction Buffer to the PCR tube, then add 1uL of the T4 DNA Ligase to the tube. | ||
+ | # Flick the tube to mix contents and spin the tube in a microcentrifuge for a few second to collect the liquid. | ||
+ | # Incubate at room temperature for 10 minutes followed by 80C for 20 minutes. This will deactivate enzymes and improve transformation efficiency. | ||
+ | # The ligation step is now completed. You may wish to store the products at -20C or begin a transformation immediately. | ||
[[Team:Wash_U/Protocol#Procedures|Back To Top]] | [[Team:Wash_U/Protocol#Procedures|Back To Top]] | ||
Revision as of 15:46, 10 July 2009