Cfrench:BALTIC: Difference between revisions
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1. Digest BioBrick U: | 1. Digest BioBrick U: | ||
38 microlitres water | *38 microlitres water | ||
2.5 microlitres DNA | *2.5 microlitres DNA | ||
5 microlitres buffer D (Promega) or 2 (New England) | *5 microlitres buffer D (Promega) or 2 (New England) | ||
1.5 microlitres SpeI | *1.5 microlitres SpeI | ||
1.5 microlitres PstI | *1.5 microlitres PstI | ||
1.5 microlitres ScaI | *1.5 microlitres ScaI | ||
TOTAL 50 microlitres | *TOTAL 50 microlitres | ||
2. Digest BioBrick D: | 2. Digest BioBrick D: | ||
38 microlitres water | *38 microlitres water | ||
2.5 microlitres DNA | *2.5 microlitres DNA | ||
5 microlitres buffer D or 2 | *5 microlitres buffer D or 2 | ||
1.5 microlitres EcoRI | *1.5 microlitres EcoRI | ||
1.5 microlitres XbaI | *1.5 microlitres XbaI | ||
1.5 microlitres ScaI | *1.5 microlitres ScaI | ||
TOTAL 50 microlitres | TOTAL 50 microlitres | ||
Line 24: | Line 24: | ||
4. Set up a ligation: | 4. Set up a ligation: | ||
4 microlitres DNA from reaction U | *4 microlitres DNA from reaction U | ||
4 microlitres DNA from reaction D | *4 microlitres DNA from reaction D | ||
1 microlitre ligase buffer | *1 microlitre ligase buffer | ||
1 microlitre T4 DNA ligase | *1 microlitre T4 DNA ligase | ||
TOTAL 10 microlitres | TOTAL 10 microlitres | ||
Revision as of 02:57, 19 February 2008
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.