HomeFAQsI am using Ph.D.™ Phage Display and the phage DNA templates do not yield readable sequence.
FAQ: I am using Ph.D.™ Phage Display and the phage DNA templates do not yield readable sequence.
The sequencing template purification protocol in the manual should provide single-stranded template of sufficient purity for dideoxy sequencing with Sequenase™ (GE Healthcare), or automated cycle sequencing with dyelabeled terminators (ABI). The procedure should be followed exactly as described in the manual: prolonged ethanol precipitation, precipitation at 20° C or centrifugation longer than 10 minutes will result in co-precipitation of salt and phage proteins, which will inhibit sequencing. Additionally, it is crucial that the phage pellet is thoroughly suspended in the iodide buffer prior to adding ethanol. If problems persist, or if another sequencing method is used, a phenol:chloroform extraction step can be added: Following suspension in Iodide Buffer, add 2 volumes of TE, extract once with phenol:chloroform (1:1) and once with chloroform, and ethanol precipitate. 5 µL of suspended template (approximately 0.5 µg) should be sufficient for sequencing; quantitation should be confirmed by agarose gel electrophoresis using 0.5 µg single stranded M13 DNA (NEB #N4040S) as a standard.
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