Cfrench:BALTIC

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BALTIC: BioBrick Assembly by Ligation of Triple-digested Components
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==BALTIC: BioBrick Assembly by Ligation of Triple-digested Components==
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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:
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1. Digest BioBrick U:
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38 microlitres water
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2.5 microlitres DNA
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5 microlitres buffer D (Promega) or 2 (New England)
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1.5 microlitres SpeI
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1.5 microlitres PstI
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1.5 microlitres ScaI
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TOTAL 50 microlitres
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2. Digest BioBrick D:
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38 microlitres water
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2.5 microlitres DNA
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5 microlitres buffer D or 2
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1.5 microlitres EcoRI
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1.5 microlitres XbaI
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1.5 microlitres ScaI
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TOTAL 50 microlitres
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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.
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4. Set up a ligation:
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4 microlitres DNA from reaction U
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4 microlitres DNA from reaction D
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1 microlitre ligase buffer
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1 microlitre T4 DNA ligase
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TOTAL 10 microlitres
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Incubate at 16 C overnight.
[[French_Lab|return to main page]]
[[French_Lab|return to main page]]

Revision as of 05:56, 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.

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