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 SfiI; 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: 38 microlitres water 2.5 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: 38 microlitres water 2.5 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.