I ligated my insert into the between of Xho I and Nde I of pet28a vector (see image), and I am wonderfing if T7 promoter or T7 terminator should be chosen as the primer for sequencing. Could you please tell me which way to go. Thank you ~
Both will work, but T7 promoter is widely used compared to T7 terminator primer.
Hi there,
These universal primer sets are designed for PCR screening and sequencing purposes. The only condition to use a defined primer for sequencing is that is should anneal around 50 bases upstream the sequence you intend to read (efficient reading starts actually around 50 bases dowstream the primer annealing region).
For forward sequencing reaction you can use T7 promoter primer and for reverse reaction you can use T7 terminator primer. Both are universal primers.
Hi H.H Nguyen,
Depending on the size of your insert (especially if it is more than 1000 bp), I'd recommend you to use both primers. Sanger sequencing can give a read length up to 1000 pb, but even so, it could be a little difficult to interpret the last nucleotides of the electropherogram.
By sequencing from both sides you will obtain the complete sequence and a more reliable result.
Hope this can help you.
Regards.
Samuel
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