How to sequence cosmid DNA insert - (Sep/19/2008 )
I have used a cosmid library to complement a phenotype in Rhizobium. I know the vector sequence and what I would like to do is PCR a bit of each end of the DNA insert so I can compare it to the sequenced genome to determine what fragment has been inserted. I only need enough sequenced on either end of the insert (~30 kb) to search the genome sequence. How can I go about getting some relatively short products to send to the sequencing facility?
If you know what the vector sequence is, then you should find out how the inserts were cloned into the cosmid vector. Pick sequences near the insert site and start sequencing from there.
I agree with smu2 -- why not design primers to the (known) vector sequence about 50 bases from the start of the cloned insert, and sequence the clone directly from both ends? You won't get 30 kb, but you will get runs showing both ends of the insert, and you can use these to design insert-specific primers if you need to PCR up a product, or just continue sequencing from ether side.