Annealing complementary primers: Difference between revisions
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''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 - | ||
#[[BioBricks construction tutorial]] | #[[BioBricks construction tutorial]] | ||
#[[Synthetic Biology:BioBricks/3A assembly | 3A assembly]] | #[[Synthetic Biology:BioBricks/3A assembly | 3A assembly]] | ||
#[[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> | |||
[[Koch Lab:Protocols/Oligonucleotide Annealing/Duplexes|Koch Lab]] (For single-molecule tethering purposes.)<br> | |||
==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. | ||
* | *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. | ||
Latest revision as of 16:36, 7 June 2008
See also Annealing primers
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 Invitrogen. Other options are described under part fabrication and annealing and primer extension. Having annealed the primers, you most likely want to use them as an insert into a 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 -
Lab Protocols
Endy:Annealing complementary primers
Silver:Annealing complementary primers
Koch Lab (For single-molecule tethering purposes.)
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
- 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.
- 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.
