Direct sequencing without cloning - how (Apr/04/2004 )

Hi!

I need your help, I'm trying to find a protocol to secuencing dsDNA without subclonning in a vector (like PGEM or PSTB1), how can I do? Because I don't want to waste time in subcloning my DNA fragments into secuencing vectors, make minipreps, etc

Thanks,

Leyre

To directly sequence your dsDNA, you need to
1) PCR amplify your target DNA and purify your PCR product, making sure you've got good amplification
2) mix one of your primers with your PCR product and send the mixture to a sequencing facility. You can sequence from both ends, in that case you need to do two sequencing reactions using each of your primers

Thanks for your answers,

but if you have to sequence a fragment from a cDNA library detected by a heterologous probe, how can I do without subclonning??