14 Polymerase Chain Reaction
217
• Extension times
An extension time of 1 min will be sufficient for a PCR product length of
up to 2 kb. For longer products the extension time may have to be
prolonged to 5 or even 10 min.
• Primer synthesis
cally,alengthofl8-28nt(10-34nt)isused.CGcontentshouldnotexceed5060%. The difference in the calculated melting points between the two primers (see above) should be ::::; 5°C.
• Primer concentration
An optimal primer concentration is in the range of0.1 to 0.5 JlM. Above this
concentration mispriming and lower yield of the specific product aretobe
expected.
• Primers for specific experimental purposes
Examples of primers for specific experimental purposes are described
briefly below together with the respective references to the literature. Mismatched primersareoften usedforsite-directedmutagenesisand toaddaS 'extension to primers which may contain additional sequence such as restrictionenzymerecognition sites, promoters, etc. When usingPCR primers
that contain mismatches it is important to note that the PCR product will
always contain the sequence of the primers rather than the template at its
ends (Figure 3 ).
IIIIIII1 = pri mer-template mismatch
Fig. 3. In regions of primer-template mismatch, primer sequence is incorporated in the PCR
product.
Also frequently used are degenerate primer mixtures for the isolation of
sequence-related novel genes. To help with the design of an optimal primer
pair for a certain sequence, computer programssuch as "TM-Oligo" are
available.
217
• Extension times
An extension time of 1 min will be sufficient for a PCR product length of
up to 2 kb. For longer products the extension time may have to be
prolonged to 5 or even 10 min.
• Primer synthesis
cally,alengthofl8-28nt(10-34nt)isused.CGcontentshouldnotexceed5060%. The difference in the calculated melting points between the two primers (see above) should be ::::; 5°C.
• Primer concentration
An optimal primer concentration is in the range of0.1 to 0.5 JlM. Above this
concentration mispriming and lower yield of the specific product aretobe
expected.
• Primers for specific experimental purposes
Examples of primers for specific experimental purposes are described
briefly below together with the respective references to the literature. Mismatched primersareoften usedforsite-directedmutagenesisand toaddaS 'extension to primers which may contain additional sequence such as restrictionenzymerecognition sites, promoters, etc. When usingPCR primers
that contain mismatches it is important to note that the PCR product will
always contain the sequence of the primers rather than the template at its
ends (Figure 3 ).
IIIIIII1 = pri mer-template mismatch
Fig. 3. In regions of primer-template mismatch, primer sequence is incorporated in the PCR
product.
Also frequently used are degenerate primer mixtures for the isolation of
sequence-related novel genes. To help with the design of an optimal primer
pair for a certain sequence, computer programssuch as "TM-Oligo" are
available.