Sequencing Primers - (Mar/22/2006 )

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Hai Friends,
I am planning to sequence my PCR products ( V. cholerae toxin genes about 600 bp). Should I use a seperate sequncing primer or the same used for the PCR reaction? What is the difference?

for sequencing PCR products, same primers can be used.

if you've cloned it in a vector, order primers that anneal 50bp away from your sequenced area.

Just as a (probably unneccassary) addition:
you will not get the sequence immediatly after your PCR primer (unless you get good sequence all the way through the 600 bp to the other side of the fragment).

So if you are for example, looking for differences between strains and the region of interest is away from the primers there would be no point in getting some additional sequencing primers (or to clone the fragments for that matted). If however, you are cloning the fragment to use for you know what, you really want to know the complete sequence of the one fragment in your plasmid clone (start to end). For this you would (probably) need the additional sequencing primers.

The most important thing is to make sure that you remove all leftover primers and primer dimers before trying to sequence the PCR fragment. The other thing too watch is don't use too much of the PCR product in the sequencing reaction - on the order of nanograms total.

Daniel

Better DNA sequencing results

[font=Garamond][size=7]

Hai Friends,
I am planning to sequence my PCR products ( V. cholerae toxin genes about 600 bp). Should I use a seperate sequncing primer or the same used for the PCR reaction? What is the difference?