Nurturing Life Sciences
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#Id: 2532
#Unit 13. Methods in Biology
In PCR, there should be no complementarity between the two primers.
#Id: 2533
The ‘Wallace rule’ (Lay Thein & Wallace 1986) is used to determine the appropriate stringency wash temperature: Tm = 4 × (number of GC base pairs) + 2 × (number of AT base pairs
The ‘Wallace rule’ (Lay Thein & Wallace 1986) is used to determine the appropriate stringency wash temperature:
Tm = 4 × (number of GC base pairs) + 2 × (number of AT base pairs
#Id: 2534
Probe length for PCR – the longer the oligonucleotide, the less chance there is of it binding to sequences other than the desired target sequence under conditions of high stringency.
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Oligonucleotide composition for PCR– the GC content will influence the stability of the resultant hybrid and hence the determination of the appropriate stringency washing conditions.
Oligonucleotide composition for PCR– the GC content will influence the stability of the resultant hybrid and hence the determination of the appropriate stringency washing conditions
.
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At the second cycle of PCR, primers will anneal to the 'long product' at sites where sequences complementary· to them are located, which will be much before the actual 3'-end of the long product.
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Extension step in the second cycle will produce a product that will be much shorter than the 'long product'; this is the 'correct' PCR product and represents the target sequence
