Posted 22 October 2001 - 09:00 PM
I'm not sure if this would definitely work in your specific case, but it should work in theory. First, design primers from the extreme 5' and 3' ends of both fragments you want to join together. Design a same (or compatible) restriction enzyme site into the ends of both the promoter 3' and gene coding region 5' primer. Make sure it is a restriction site that is not found in the sequence of the promoter or coding sequence. Now do a PCR reaction to amplify the two separate fragments. After this, you have a the promoter sequence with the restriction site at it's 3' end and in the other pcr reaction, you have the coding sequence with the restriction site at the 5' end. Now digest both with that enzyme and clean up the reaction. Then, ligate them together with t4 dna ligase. This should directionally join them together. If you want to put this whole fragment into a vector, just design differnt restriction sites into the promoter5' primer and the coding sequence 3' primer. Make sure that these are compatible with sites on your vector, but not found in you sequences. Good luck!