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Annealing complementary primers: Difference between revisions
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Annealing complementary primers (view source)
Revision as of 10:29, 11 January 2006
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==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. | ||
