Converting RE sites - (May/13/2009 )

Please help ASAP! I have a large (14kb) DNA fragment cloned into a PstI site, but I need to clone this fragment into another vector at an SpeI site. I want to use an oligo linker to change my PstI site (kill it) to an SpeI site.

Will these oligos work to change PstI to SpeI:

5- TGCA-ACTAGT-3
3-TGATCA-ACGT-5

Thanks!

PCRing it up with new set of primers with SpeI site is an option but amplifying 14kb from a vector is a daunting task.

I would like to know how you solve this problem. Please post once you are successful.

Thanks in advance.

catamere on May 13 2009, 04:52 PM said:

they won't work, you have twice the same oligo written 5-3 and 3-5. you have to reverse complement it.
But as noelmathur has suggested, it would be better to amplify by PCR the gene you want to insert, using overhanged primers that include the restriction sites of the vector.
If you PCR the complete vector, you should sequence the complete vector as there could be some mutations during PCR. This is a long task.
something like 5'-nnnnnnnnACTAGTNNNNNNNNNN-3' where nnnnn is any nucleotide and NNN is the beginning of the sequence of your insert. Do the same for the reverse primer (reverse-complement).
Do the PCR, cut and insert in your vector. (and then you should sequence the insert, as there could be some mutations during PCR.)