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DNA CONSIDERATIONS
Success has been
achieved using DNA from kits by Qiagen, Promega
and Bio 101. For optimum results for purifying PCR product before
sequencing, we recommend centricon-100 columns,Amicon
PCR Kit and Qiagen QIAquick PCR kit.
The recommended quantities
of DNA are as follows:
Phage DNA 50-100 ng -5ul /per reaction
Plasmid DNA 250-500 ng- 6ul /per reaction
All DNA should be resuspended in dH20
.
PCR
product: The
amount of template to use in a cycle sequencing reaction.
100-200 bp -1-3 ng
200-500 bp -3-10 ng
500-1000 bp -5-20 ng
1000-2000 bp -10-40 ng
If possible, quantitate the amount of purified DNA by
measuring the absorbance at 260 nm or by some other method.
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