Cfrench:BALTIC
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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 '''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. | ||
[[French_Lab|return to main page]] | [[French_Lab|return to main page]] | ||
Current revision
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.
