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BALTIC: BioBrick Assembly by Ligation of Triple-digested Components | ==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. | |||
[[French_Lab|return to main page]] | [[French_Lab|return to main page]] |
Revision as of 02:56, 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.