Preparation of sequencing primer

Preliminary screening of templates with the ddT reaction (Sanger et al., 1980) 

Recently Hindley, Porter and Smith (1981, unpublished results) have used a totally synthetic single-stranded DNA primer of 18 nucleotides equivalent to the 3 -end of the 96-nucleotide primer. This has the sequence  

3 pmol of this primer are hybridized to 1-2 pmol of the template in 

In the presence of a 2 /zM-concentration of the competing dNTP most of the termination events occur in the desired size-distribution range giving reasonably uniform band patterns. However, it should be emphasized that these relative concentrations should be determined by trial and error before serious sequencing is commenced. Note that in the case of the ddA-terminating reaction 

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