Cfrench:BALTIC - Revision history

Cfrench at 17:19, 19 February 2008

Cfrench — Tue, 19 Feb 2008 17:19:14 GMT

←Older revision		Revision as of 17:19, 19 February 2008
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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:	+	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:		1. Digest BioBrick U:

Cfrench at 17:18, 19 February 2008

Cfrench — Tue, 19 Feb 2008 17:18:47 GMT

←Older revision		Revision as of 17:18, 19 February 2008
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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:	+	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:		1. Digest BioBrick U:

Cfrench at 09:59, 19 February 2008

Cfrench — Tue, 19 Feb 2008 09:59:37 GMT

←Older revision		Revision as of 09:59, 19 February 2008
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	1. Digest BioBrick U:		1. Digest BioBrick U:
-	*38 microlitres water	+	*35.5 microlitres water
-	*2.5 microlitres DNA	+	*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
Line 13:		Line 13:

	2. Digest BioBrick D:		2. Digest BioBrick D:
-	*38 microlitres water	+	*35.5 microlitres water
-	*2.5 microlitres DNA	+	*5 microlitres DNA
	*5 microlitres buffer D or 2		*5 microlitres buffer D or 2
	*1.5 microlitres EcoRI		*1.5 microlitres EcoRI

Cfrench at 09:57, 19 February 2008

Cfrench — Tue, 19 Feb 2008 09:57:47 GMT

←Older revision		Revision as of 09:57, 19 February 2008
Line 4:		Line 4:

	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

Cfrench at 09:56, 19 February 2008

Cfrench — Tue, 19 Feb 2008 09:56:45 GMT

←Older revision		Revision as of 09:56, 19 February 2008
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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.

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Cfrench: New page: BALTIC: BioBrick Assembly by Ligation of Triple-digested Components return to main page

Cfrench — Tue, 19 Feb 2008 09:46:22 GMT

New page: BALTIC: BioBrick Assembly by Ligation of Triple-digested Components return to main page

New page

BALTIC: BioBrick Assembly by Ligation of Triple-digested Components

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