sequencing a restriction fragment - (Mar/29/2006 )

Hi all,

I have a not1 fragment in a plasmid of which the sequence is not known. I would like to make a caseette of this fragment to clone it into another plasmid and also add RE sites to the cassette. How do i do that without knowing the sequence to design primers. Is there any way to clone a restriction fragment and sequence it, all systems like TA and TOPO seem to be meant for PCR product sequencing
utterly lost!

absolutely!

1. clone your fragment into a commercial vector (Not I or isoschizomer; or blunt the ends)
2. send off for sequencing

the trick is...knowing your commercial vector. there are many readily available sequencing primers based solely on vector sequence. you may have to check that a vector has sequencing primers available before you start

otherwise, how would we ever get the sequence of unknown gDNA fragments?

Digest your plasmid with NotI, recover the fragment, and clone it into a NotI digested vector for which the sequence is known. Use two vector-borne primers to sequence the insert, and use that information to design insert-specific primers with whatever 5' modifications you desire.

[edit] Whoops - I cross-posted with aimikins, but at least we both gave you the same answer... [/edit]

Homebrew...damn we're good

Homebrew...damn we're good