plasmid sequencing - (Apr/21/2006 )

Hi,

Why are two primers needed for sequencing the insert within a vector plasmid?

I was told by colleges that the ends and the beginning of the sequencing reaction is not usually good and this is to make sure the whole insert is sequenced. But I don't understand as I am using two primers right at the ends of my insert.

And why are the begining and the end of sequencing reaction not good?

Thanks so much in advance
Perl

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The first 20 peaks or so on the electrpherogram aren't interpretable, don't know the reason behind this though. And after severall hundred peaks, the distance between the peaks becomes bigger and it becomes harder to interpret your data. We mostly go up to 800 bp or something (also depending on template quality/quantity).

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Thanks for the reply

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Hi.

Whether you use manual sequencing or automated sequencing, the first few dideoxyDNA products are bad because they are very small and ofetn don't resolve well. That is why the sequencing primers are usually set well away from the cloning region. It takes about 40nt for polyacrylamide to settle down and be able to resolve single nt additions.

At the other end of the reaction, if the insert gets too big, automated systems have a problem because of mass differences between the four fluorescent probes. If you look at an electropherogram, you see that the spacing between bases gets smaller, and you start getting more overlap. This means that the software has to do more calculations per base, in order to decide which base to call.

Even if your insert isn't too big, it's usually a good idea to sequence both strands, so you can use one strand to check the other. An "N" call on one strand is unlikely to be an "N" on the other. If it is, then there are different issues to deal with, such as polymorphisms. It's also good to be able to read through to the other side of the plasmid cloning site.

Hope this makes things a bit clearer.

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Hi,

Thanks a lot. I am begining to understand now!

Perl

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