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Annealing complementary primers: Difference between revisions
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*The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind. | *The salt shields the negative charges on the single-stranded DNA molecules, allowing them to come close enough to bind. | ||
*Anneal the primers by heating them at least 5°C above their melting point and cooling them down slowly in stages using a [[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. | ||
*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 using adding the 5' phosphate using a protocol such as [[PNK Treatment of DNA Ends]] | ||
