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From 2014.igem.org

Contents 1 BioBrick Assembly 1.1 Procedure 1.1.1 Enzymes 1.1.2 Digest reaction 1.1.3 Segregate process 1.1.4 DNA extration from agarose gels 1.1.5 Ligation

BioBrick Assembly

This protocol is based on the original [http://parts.igem.org/Help:Assembly/3A_Assembly 3A Assembly] from iGEM. We modified it to a assembly that places the core of one BioBrick - here called Part A - into another BioBrick - here called Part B.

TODO: Process illustration [Format I and Format II]

Procedure

Enzymes

Note: Manipulate the enzymes with a proper subzero temperature support.

Part A:

• Enzyme A: EcoRI
• Enzyme B: SpeI

Part B with Format I (Part B placed after Part A):

• Enzyme A: EcoRI
• Enzyme B: XbaI

Part B with Format II (Part B placed before Part A):

• Enzyme A: SpeI
• Enzyme B: PstI

Digest reaction

1. Take a 1.5ml microcentrifuge tube (Eppendorf) and put:
   1. xμl of H₂0 milliQ (complement)
   2. 5μl of buffer (Fast Digest Buffer 10x)
   3. xμl of Part A DNA
   4. 1μl of Enzyme A
   5. 1μl of Enzyme B
2. Repeat step 1 with Part B
3. Mix gently both tubes
4. Incubate at 37°C for one hour
5. Store Part B at -20°C.

Segregate process

1. part A
2. big box, gel 0.8% agarose (and BET concentration???????)
3. 1 hour
4. minimal exposure of UV light
5. mass of the ependorf (before / after)

DNA extration from agarose gels

Ligation