Cfrench:BALTIC

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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 04:57, 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.

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