IGEM:Brown/2007/Lab Protocols/Constructing a Plasmid

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Constructing a Plasmid

Set up the digests

1. For backbone cut 5 ug of plasmid DNA in a 50 ul reaction:

 -5 ug of backbone DNA
 -5 ul of 10X buffer of choice
 -(5 ul of 10X BSA, optional)
 -1-2 ul of enzume depending on the unit concentration
 - up to 50 ul with autoclaved miliQ water

2. For insert, cut enough plasmid to yield approximately 5 ug of insert. If your plasmid is 10 kb and your insert will be 1.5 kb, cutting 30 ug will yield 4.5 ug of insert.

 -ug of insert DNA
 -5 ul of 10X buffer of choice
 -(5 ul of 10X BSA, optional)
 -1 unit of enzyme per ug of DNA
 -up to 50 ul with autoclaved miliQ water.

As a general rule, 1 unit of enzyme will cut 1 ug of plasmid DNA in 1 hour, so you can adjust the units of enzyme according to how much DNA you have and how long your digest will last.

3. Digest at the required temperature (usually 37 C) for 2 hours to overnight.

4. Check the 2 ul of each digest on an agarose gel to make sure there is complete cutting, photograph.

5. If the backbone is cut with only one enzyme, phosphatase the backbone by adding .5 ul of calf intestinal phosphatase (CIP) and incubate for 30 minutes at 37 C.

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