Cfrench:BALTIC

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(New page: BALTIC: BioBrick Assembly by Ligation of Triple-digested Components return to main page)
Current revision (13:19, 19 February 2008) (view source)
 
(4 intermediate revisions not shown.)
Line 1: Line 1:
-
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.

return to main page

Personal tools