Richard Lab:Amplified insert assembly - Revision history

Tlr20 at 03:48, 23 April 2012

2012-04-23T03:48:12Z

←Older revision		Revision as of 03:48, 23 April 2012
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	==References==		==References==
-	*Speer, M.A. and T.L. Richard. 2011. '''Amplified insert assembly: an optimized approach to standard assembly of BioBrick™ genetic circuits''' ''Journal of Biological Engineering'' 5:17	+	*Speer, M.A. and T.L. Richard. 2011. [http://www.jbioleng.org/content/5/1/17 '''Amplified insert assembly: an optimized approach to standard assembly of BioBrick™ genetic circuits''' ''Journal of Biological Engineering'' 5:17]

	==Contact==		==Contact==

Tlr20 at 03:43, 25 February 2012

2012-02-25T03:43:04Z

←Older revision		Revision as of 03:43, 25 February 2012
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	<center>'''Amplified Insert Assembly'''</center>		<center>'''Amplified Insert Assembly'''</center>
	==Overview==		==Overview==
-	This is a lab specific protocol. For more information on the benefits of Amplified Insert Assembly see the [[Amplified Insert Assembly\|consensus protocol]]. A [http://www.youtube.ug/watch?v=Tvxx2bL0R2I&context=C3df62b2ADOEgsToPDskIOrrbFVhSy4joiAlQXxIYp ~~U-tube~~ video summary of the method] ~~is also available~~.	+	This is a lab specific protocol. For more information on the benefits of Amplified Insert Assembly see the [[Amplified Insert Assembly\|consensus protocol]]. A You-Tube [http://www.youtube.ug/watch?v=Tvxx2bL0R2I&context=C3df62b2ADOEgsToPDskIOrrbFVhSy4joiAlQXxIYp video summary of the Amplified Insert Assembly method] provides a quick and entertaining visual introduction.

	This protocol is typically used to do bio-brick assembly with restriction sites consisting of the following configuration:		This protocol is typically used to do bio-brick assembly with restriction sites consisting of the following configuration:

Tlr20 at 03:33, 25 February 2012

2012-02-25T03:33:33Z

←Older revision		Revision as of 03:33, 25 February 2012
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	<center>'''Amplified Insert Assembly'''</center>		<center>'''Amplified Insert Assembly'''</center>
	==Overview==		==Overview==
-	This is a lab specific protocol. For more information on the benefits of Amplified Insert Assembly see the [[Amplified Insert Assembly\|consensus protocol]].	+	This is a lab specific protocol. For more information on the benefits of Amplified Insert Assembly see the [[Amplified Insert Assembly\|consensus protocol]]. A [http://www.youtube.ug/watch?v=Tvxx2bL0R2I&context=C3df62b2ADOEgsToPDskIOrrbFVhSy4joiAlQXxIYp U-tube video summary of the method] is also available.

	This protocol is typically used to do bio-brick assembly with restriction sites consisting of the following configuration:		This protocol is typically used to do bio-brick assembly with restriction sites consisting of the following configuration:

Michael A. Speer: /* References */

2011-12-17T20:50:31Z

References

←Older revision		Revision as of 20:50, 17 December 2011
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	==References==		==References==
-	Speer, M.A. and T.L. Richard. 2011. '''Amplified insert assembly: an optimized approach to standard assembly of BioBrick™ genetic circuits''' ''Journal of Biological Engineering'' 5:17	+	*Speer, M.A. and T.L. Richard. 2011. '''Amplified insert assembly: an optimized approach to standard assembly of BioBrick™ genetic circuits''' ''Journal of Biological Engineering'' 5:17

	==Contact==		==Contact==

Michael A. Speer: /* References */

2011-12-17T20:49:50Z

References

←Older revision		Revision as of 20:49, 17 December 2011
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	==References==		==References==
-	~~Michael~~ A. ~~Speer~~ and ~~Tom~~ L. Richard. 2011. '''Amplified insert assembly: an optimized approach to standard assembly of BioBrick™ genetic circuits''' ''Journal of Biological Engineering'' 5:17	+	Speer, M.A. and T.L. Richard. 2011. '''Amplified insert assembly: an optimized approach to standard assembly of BioBrick™ genetic circuits''' ''Journal of Biological Engineering'' 5:17

	==Contact==		==Contact==

Michael A. Speer: /* References */

2011-12-17T20:49:08Z

References

←Older revision		Revision as of 20:49, 17 December 2011
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	==References==		==References==
-	Michael A. Speer and Tom L. Richard. 2011. Amplified insert assembly: an optimized approach to standard assembly of BioBrick™ genetic circuits. ''Journal of Biological Engineering'' 5:17	+	Michael A. Speer and Tom L. Richard. 2011. '''Amplified insert assembly: an optimized approach to standard assembly of BioBrick™ genetic circuits''' ''Journal of Biological Engineering'' 5:17

	==Contact==		==Contact==

Michael A. Speer at 20:48, 17 December 2011

2011-12-17T20:48:15Z

←Older revision		Revision as of 20:48, 17 December 2011
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	;;#This is why we use a high-fidelity polymerase		;;#This is why we use a high-fidelity polymerase
	::#We're sequencing the constructs anyway so we'd spot any mutations.		::#We're sequencing the constructs anyway so we'd spot any mutations.
		+
		+	==References==
		+	Michael A. Speer and Tom L. Richard. 2011. Amplified insert assembly: an optimized approach to standard assembly of BioBrick™ genetic circuits. ''Journal of Biological Engineering'' 5:17

	==Contact==		==Contact==

Michael A. Speer: /* Procedure */

2011-04-12T19:19:43Z

Procedure

←Older revision		Revision as of 19:19, 12 April 2011
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	::*Only run 25-30 cycles as this will help ensure high fidelity.<br>		::*Only run 25-30 cycles as this will help ensure high fidelity.<br>
	3. Start [[Richard Lab:Restriction Digest\|Digesting]] the "vector" while the PCR is cycling.<br>		3. Start [[Richard Lab:Restriction Digest\|Digesting]] the "vector" while the PCR is cycling.<br>
-	::*For help on deciding which enzymes to use see [[Enzyme selection for BioBricks digest\|this ~~link~~]].	+	::*For help on deciding which enzymes to use see [[Enzyme selection for BioBricks digest\|this page]].
	4. Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids\|this protocol]].<br>		4. Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids\|this protocol]].<br>
	5. [[Richard Lab:Restriction Digest\|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).<br>		5. [[Richard Lab:Restriction Digest\|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).<br>

Michael A. Speer: /* Procedure */

2011-04-12T19:19:18Z

Procedure

←Older revision		Revision as of 19:19, 12 April 2011
Line 20:		Line 20:
	::*Only run 25-30 cycles as this will help ensure high fidelity.<br>		::*Only run 25-30 cycles as this will help ensure high fidelity.<br>
	3. Start [[Richard Lab:Restriction Digest\|Digesting]] the "vector" while the PCR is cycling.<br>		3. Start [[Richard Lab:Restriction Digest\|Digesting]] the "vector" while the PCR is cycling.<br>
		+	::*For help on deciding which enzymes to use see [[Enzyme selection for BioBricks digest\|this link]].
	4. Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids\|this protocol]].<br>		4. Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids\|this protocol]].<br>
	5. [[Richard Lab:Restriction Digest\|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).<br>		5. [[Richard Lab:Restriction Digest\|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).<br>

Michael A. Speer: /* Procedure */

2011-04-06T19:27:12Z

Procedure

←Older revision		Revision as of 19:27, 6 April 2011
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	::*Use the same primers you use for colony PCR (Annealing Temp of 55-60°C).<br>		::*Use the same primers you use for colony PCR (Annealing Temp of 55-60°C).<br>
	::*Only run 25-30 cycles as this will help ensure high fidelity.<br>		::*Only run 25-30 cycles as this will help ensure high fidelity.<br>
-	3. [[Richard Lab:Restriction Digest\|~~Digest~~]] the "vector" ~~for 2 hours~~.<br>	+	3. Start [[Richard Lab:Restriction Digest\|Digesting]] the "vector" while the PCR is cycling.<br>
	4. Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids\|this protocol]].<br>		4. Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids\|this protocol]].<br>
	5. [[Richard Lab:Restriction Digest\|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).<br>		5. [[Richard Lab:Restriction Digest\|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).<br>
-	6. ~~Add~~ 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done.<br>	+	6. Once the insert is digesting add 1μL Antarctic Phosphatase and 6μL AP Buffer to the "vector" digest and incubate until the "insert" digest is done.<br>
	7. Kill all reactions by incubating for 20 mins at 80°C.<br>		7. Kill all reactions by incubating for 20 mins at 80°C.<br>
	8. [[Richard Lab:Ligation\|Ligate]] at a molar ratio of 4:1 (insert:vector).<br>		8. [[Richard Lab:Ligation\|Ligate]] at a molar ratio of 4:1 (insert:vector).<br>

←Older revision		Revision as of 19:19, 12 April 2011
Line 20:		Line 20:
	::*Only run 25-30 cycles as this will help ensure high fidelity.<br>		::*Only run 25-30 cycles as this will help ensure high fidelity.<br>
	3. Start [[Richard Lab:Restriction Digest\|Digesting]] the "vector" while the PCR is cycling.<br>		3. Start [[Richard Lab:Restriction Digest\|Digesting]] the "vector" while the PCR is cycling.<br>
-	::*For help on deciding which enzymes to use see [[Enzyme selection for BioBricks digest\|this ~~link~~]].	+	::*For help on deciding which enzymes to use see [[Enzyme selection for BioBricks digest\|this page]].
	4. Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids\|this protocol]].<br>		4. Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids\|this protocol]].<br>
	5. [[Richard Lab:Restriction Digest\|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).<br>		5. [[Richard Lab:Restriction Digest\|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).<br>

←Older revision		Revision as of 19:19, 12 April 2011
Line 20:		Line 20:
	::*Only run 25-30 cycles as this will help ensure high fidelity.<br>		::*Only run 25-30 cycles as this will help ensure high fidelity.<br>
	3. Start [[Richard Lab:Restriction Digest\|Digesting]] the "vector" while the PCR is cycling.<br>		3. Start [[Richard Lab:Restriction Digest\|Digesting]] the "vector" while the PCR is cycling.<br>
		+	::*For help on deciding which enzymes to use see [[Enzyme selection for BioBricks digest\|this link]].
	4. Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids\|this protocol]].<br>		4. Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids\|this protocol]].<br>
	5. [[Richard Lab:Restriction Digest\|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).<br>		5. [[Richard Lab:Restriction Digest\|Digest]] insert for 1 hour (adding DpnI along with the other restriction endonucleases).<br>