Annealing complementary primers - Revision history

Steven J. Koch: /* Lab Protocols */

2008-06-07T23:36:40Z

Lab Protocols

←Older revision		Revision as of 23:36, 7 June 2008
Line 10:		Line 10:
	[[Endy:Annealing complementary primers]]<br>		[[Endy:Annealing complementary primers]]<br>
	[[Silver: Oligonucleotide Inserts\|Silver:Annealing complementary primers]]<br>		[[Silver: Oligonucleotide Inserts\|Silver:Annealing complementary primers]]<br>
		+	[[Koch Lab:Protocols/Oligonucleotide Annealing/Duplexes\|Koch Lab]] (For single-molecule tethering purposes.)<br>

	==Notes==		==Notes==

Reshma P. Shetty at 21:31, 9 October 2007

2007-10-09T21:31:16Z

←Older revision		Revision as of 21:31, 9 October 2007
Line 10:		Line 10:
	[[Endy:Annealing complementary primers]]<br>		[[Endy:Annealing complementary primers]]<br>
	[[Silver: Oligonucleotide Inserts\|Silver:Annealing complementary primers]]<br>		[[Silver: Oligonucleotide Inserts\|Silver:Annealing complementary primers]]<br>
-

	==Notes==		==Notes==

Reshma P. Shetty at 21:31, 9 October 2007

2007-10-09T21:31:06Z

←Older revision		Revision as of 21:31, 9 October 2007
Line 1:		Line 1:
-	''See also [[Annealing ~~Primers~~]]''	+	''See also [[Annealing primers]]''
		+
	==Introduction==		==Introduction==
	A simple and cheap way to make a short (< 100 bp) piece of DNA is to order two complementary primers from a company such as [http://www.invitrogen.com Invitrogen]. Other options are described under [[Synthetic Biology:BioBricks/Part fabrication \| part fabrication]] and [[Annealing and primer extension\|annealing and primer extension]]. Having annealed the primers, you most likely want to use them as an insert into a [[Synthetic Biology:Vectors \| vector]] (the linked page describes BioBrick vectors). Here are some pages that describe methods to assemble two parts if they correspond to a BioBricks standard -		A simple and cheap way to make a short (< 100 bp) piece of DNA is to order two complementary primers from a company such as [http://www.invitrogen.com Invitrogen]. Other options are described under [[Synthetic Biology:BioBricks/Part fabrication \| part fabrication]] and [[Annealing and primer extension\|annealing and primer extension]]. Having annealed the primers, you most likely want to use them as an insert into a [[Synthetic Biology:Vectors \| vector]] (the linked page describes BioBrick vectors). Here are some pages that describe methods to assemble two parts if they correspond to a BioBricks standard -

Barry Canton at 16:37, 30 January 2006

2006-01-30T16:37:27Z

←Older revision		Revision as of 16:37, 30 January 2006
Line 1:		Line 1:
		+	''See also [[Annealing Primers]]''
	==Introduction==		==Introduction==
	A simple and cheap way to make a short (< 100 bp) piece of DNA is to order two complementary primers from a company such as [http://www.invitrogen.com Invitrogen]. Other options are described under [[Synthetic Biology:BioBricks/Part fabrication \| part fabrication]] and [[Annealing and primer extension\|annealing and primer extension]]. Having annealed the primers, you most likely want to use them as an insert into a [[Synthetic Biology:Vectors \| vector]] (the linked page describes BioBrick vectors). Here are some pages that describe methods to assemble two parts if they correspond to a BioBricks standard -		A simple and cheap way to make a short (< 100 bp) piece of DNA is to order two complementary primers from a company such as [http://www.invitrogen.com Invitrogen]. Other options are described under [[Synthetic Biology:BioBricks/Part fabrication \| part fabrication]] and [[Annealing and primer extension\|annealing and primer extension]]. Having annealed the primers, you most likely want to use them as an insert into a [[Synthetic Biology:Vectors \| vector]] (the linked page describes BioBrick vectors). Here are some pages that describe methods to assemble two parts if they correspond to a BioBricks standard -

Barry Canton at 16:35, 30 January 2006

2006-01-30T16:35:40Z

←Older revision		Revision as of 16:35, 30 January 2006
Line 8:		Line 8:
	[[Endy:Annealing complementary primers]]<br>		[[Endy:Annealing complementary primers]]<br>
	[[Silver: Oligonucleotide Inserts\|Silver:Annealing complementary primers]]<br>		[[Silver: Oligonucleotide Inserts\|Silver:Annealing complementary primers]]<br>
-	~~==Method==~~

-	~~===Primer reconstitution===~~
-	~~When the primers arrive, redissolve them in 50 mM Tris buffer to yield a concentration of ~800 ng/μl. See this page on [[Reconstituting primers \| reconstituting primers]] for more information.~~
-
-	~~===Annealing Mix===~~
-	*For the annealing mix one recipe that works is as follows -
-	**8 μL of each of the concentrated primers.
-	**4 μL of salt solution (10 mM NaCl)
-	**20 μL of water
-
-	~~===Option 1===~~
-	Anneal the primers by heating them at least 5°C above their melting point and cooling them down slowly in stages using a [[Endy:Thermocyclers\|Thermocycler]]. Melting temperature calculations can best be done using software such as [[VectorNTI]] or data may come with the primers themselves.
-
-	~~===Option 2===~~
-	A simpler approach is to add the above mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally. Most primers pairs with length less than 100bp should be fully melted at 100<sup>o</sup>C and certainly any non-specific binding should be melted at that temperature.

	==Notes==		==Notes==

Barry Canton at 16:33, 30 January 2006

2006-01-30T16:33:33Z

←Older revision		Revision as of 16:33, 30 January 2006
Line 5:		Line 5:
	#[[Silver: BB Strategy \| Silver lab strategy]].		#[[Silver: BB Strategy \| Silver lab strategy]].

		+	==Lab Protocols==
		+	[[Endy:Annealing complementary primers]]<br>
		+	[[Silver: Oligonucleotide Inserts\|Silver:Annealing complementary primers]]<br>
	==Method==		==Method==

Barry Canton at 17:29, 11 January 2006

2006-01-11T17:29:11Z

←Older revision		Revision as of 17:29, 11 January 2006
Line 24:		Line 24:
	==Notes==		==Notes==
	*Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by adding the 5' phosphate using a protocol such as [[PNK Treatment of DNA Ends]]		*Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by adding the 5' phosphate using a protocol such as [[PNK Treatment of DNA Ends]]
		+
		+	*The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind. The salt concentration listed above is designed to give an excess of positive sodium ions over negatively charged DNA bases.

	*As the paired oligos cool, they will also form multimers of your insert. To release them, you should heat the mixture of your vector and insert DNA to about 65C and let it cool prior to adding ligase.		*As the paired oligos cool, they will also form multimers of your insert. To release them, you should heat the mixture of your vector and insert DNA to about 65C and let it cool prior to adding ligase.

-	*~~The salt shields~~ the ~~negative charges on the single-stranded DNA molecules~~, ~~allowing them~~ to ~~come~~ close ~~enough~~ to ~~bind~~. ~~The salt concentration listed above is designed to give an excess~~ of ~~positive sodium ions over negatively charged DNA bases~~.	+	*If you will subsequently be ligating the annealed primers into a vector, it is possible to add the vector into the annealing mix and then add ligase buffer and ligase once the mix gets close to room temperature. this should reduce the likelihood of insert multimers forming.

Barry Canton: /* Introduction */

2006-01-10T22:02:14Z

Introduction

←Older revision		Revision as of 22:02, 10 January 2006
Line 1:		Line 1:
	==Introduction==		==Introduction==
-	A simple and cheap way to make a short (< 100 bp) piece of DNA is to order two complementary primers from a company such as [http://www.invitrogen.com Invitrogen]. Other options are described under [[Synthetic Biology:BioBricks/Part fabrication \| part fabrication]] and [[Annealing and primer extension\|annealing and primer extension]]. Having annealed the primers, you most likely want to use them as an insert into a [[Synthetic Biology:Vectors \| vector]] (the linked page describes BioBrick vectors). Here are some pages that describe methods to assemble two parts if they correspond to a BioBricks standard -	+	A simple and cheap way to make a short (< 100 bp) piece of DNA is to order two complementary primers from a company such as [http://www.invitrogen.com Invitrogen]. Other options are described under [[Synthetic Biology:BioBricks/Part fabrication \| part fabrication]] and [[Annealing and primer extension\|annealing and primer extension]]. Having annealed the primers, you most likely want to use them as an insert into a [[Synthetic Biology:Vectors \| vector]] (the linked page describes BioBrick vectors). Here are some pages that describe methods to assemble two parts if they correspond to a BioBricks standard -
	#[[BioBricks construction tutorial]]		#[[BioBricks construction tutorial]]
	#[[Synthetic Biology:BioBricks/3A assembly \| 3A assembly]]		#[[Synthetic Biology:BioBricks/3A assembly \| 3A assembly]]

Barry Canton at 22:02, 10 January 2006

2006-01-10T22:02:00Z

←Older revision		Revision as of 22:02, 10 January 2006
Line 1:		Line 1:
-	A simple and cheap way to make a short (< 100 bp) piece of DNA is to order two complementary primers from a company such as [http://www.invitrogen.com Invitrogen].	+	==Introduction==
		+	A simple and cheap way to make a short (< 100 bp) piece of DNA is to order two complementary primers from a company such as [http://www.invitrogen.com Invitrogen]. Other options are described under [[Synthetic Biology:BioBricks/Part fabrication \| part fabrication]] and [[Annealing and primer extension\|annealing and primer extension]]. Having annealed the primers, you most likely want to use them as an insert into a [[Synthetic Biology:Vectors \| vector]] (the linked page describes BioBrick vectors). Here are some pages that describe methods to assemble two parts if they correspond to a BioBricks standard -
		+	#[[BioBricks construction tutorial]]
		+	#[[Synthetic Biology:BioBricks/3A assembly \| 3A assembly]]
		+	#[[Silver: BB Strategy \| Silver lab strategy]].

-	*When the primers arrive, redissolve them in 50 mM Tris buffer to yield a concentration of ~800 ng/μl.	+	==Method==

		+	===Primer reconstitution===
		+	When the primers arrive, redissolve them in 50 mM Tris buffer to yield a concentration of ~800 ng/μl. See this page on [[Reconstituting primers \| reconstituting primers]] for more information.
		+
		+	===Annealing Mix===
	*For the annealing mix one recipe that works is as follows -		*For the annealing mix one recipe that works is as follows -
	**8 μL of each of the concentrated primers.		**8 μL of each of the concentrated primers.
Line 8:		Line 16:
	**20 μL of water		**20 μL of water

-	*The salt shields the ~~negative charges on the single-stranded DNA molecules, allowing~~ them to come ~~close enough to bind~~.	+	===Option 1===
		+	Anneal the primers by heating them at least 5°C above their melting point and cooling them down slowly in stages using a [[Endy:Thermocyclers\|Thermocycler]]. Melting temperature calculations can best be done using software such as [[VectorNTI]] or data may come with the primers themselves.

-	*Anneal the primers by heating them at least 5°C above their melting point and cooling them down slowly in stages using a [[Endy:Thermocyclers\|Thermocycler]]. Melting temperature calculations can best be done using software such as [[VectorNTI]] or data may come with the primers themselves.	+	===Option 2===
-		+	A simpler approach is to add the above mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally. Most primers pairs with length less than 100bp should be fully melted at 100<sup>o</sup>C and certainly any non-specific binding should be melted at that temperature.
-	*A simpler approach is to add the above mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally. Most primers pairs with length less than 100bp should be fully melted at 100<sup>o</sup>C and certainly any non-specific binding should be melted at that temperature.	+

		+	==Notes==
	*Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by adding the 5' phosphate using a protocol such as [[PNK Treatment of DNA Ends]]		*Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by adding the 5' phosphate using a protocol such as [[PNK Treatment of DNA Ends]]
		+
		+	*As the paired oligos cool, they will also form multimers of your insert. To release them, you should heat the mixture of your vector and insert DNA to about 65C and let it cool prior to adding ligase.
		+
		+	*The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind. The salt concentration listed above is designed to give an excess of positive sodium ions over negatively charged DNA bases.

Barry Canton at 15:06, 6 January 2006

2006-01-06T15:06:08Z

←Older revision		Revision as of 15:06, 6 January 2006
Line 14:		Line 14:
	*A simpler approach is to add the above mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally. Most primers pairs with length less than 100bp should be fully melted at 100<sup>o</sup>C and certainly any non-specific binding should be melted at that temperature.		*A simpler approach is to add the above mix in a PCR tube to a beaker of boiling water and just allow the water to cool down naturally. Most primers pairs with length less than 100bp should be fully melted at 100<sup>o</sup>C and certainly any non-specific binding should be melted at that temperature.

-	*Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by ~~using~~ adding the 5' phosphate using a protocol such as [[PNK Treatment of DNA Ends]]	+	*Unless you have ordered your primers with 5' phosphate added you will probably improve the efficiency of any subsequent cloning steps by adding the 5' phosphate using a protocol such as [[PNK Treatment of DNA Ends]]