Cfrench:BALTIC

BALTIC: BioBrick Assembly by Ligation of Triple-digested Components

This is a method for assembling two BioBricks, U (upstream) and D (downstream), without purifying fragments from a gel or using alternate vectors with different antibiotic resistance elements. It relies on digestion with a third enzyme cutting within the vector, so that each BioBrick is attached to a partial vector. The third enzyme can be ScaI, which cuts within the beta-lactamase gene of ampicillin-resistant vectors, or alternatively PvuI or SspI; these enzymes will all work with pSB1A2 or pSB1A3. Obviously, the restriction site chosen must be absent from the sequence of both BioBricks. The protocol is as follows:

1. Digest BioBrick U:

• 35.5 microlitres water
• 5 microlitres DNA
• 5 microlitres buffer D (Promega) or 2 (New England)
• 1.5 microlitres SpeI
• 1.5 microlitres PstI
• 1.5 microlitres ScaI
• TOTAL 50 microlitres

2. Digest BioBrick D:

• 35.5 microlitres water
• 5 microlitres DNA
• 5 microlitres buffer D or 2
• 1.5 microlitres EcoRI
• 1.5 microlitres XbaI
• 1.5 microlitres ScaI

TOTAL 50 microlitres

3. After several hours at 37 C, purify the DNA from both reactions using 5 microlitres of glass beads and elute to 10 microlitres of EB.

4. Set up a ligation:

• 4 microlitres DNA from reaction U
• 4 microlitres DNA from reaction D
• 1 microlitre ligase buffer
• 1 microlitre T4 DNA ligase

TOTAL 10 microlitres

Incubate at 16 C overnight.

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