Inserting b2AA9 into pGHE

Inserting b2AR 9 aa (b2AA9) into TAAR1 pGHE (phenol chloroform cleanups and other treatments omitted)

1. Use PCR to amplify 9aa region and insert AflII (5’) and AvrII (3’) cutting sites.

2. Use PCR to amplify TAAR1 out of pGHE and introduce AvrII (5’) cutting site right upstream of coding sequence. The reverse primer should bind downstream of the SacII (3’) cutting site to obtain TAAR1 coding sequence with the AvrII and SacII cutting sites at their corresponding ends.

3. Use PCR to create an AflII cutting site between the Xenopus 5’ UTR and TAAR1 coding sequence in TAAR1 pGHE.

4. Digest b2AA9 PCR product with AflII and AvrII.

5. Digest TAAR1 PCR produce with AvrII and SacII.

6. Digest TAAR1 pGHE with AflII (5’) and SacII (3’).

7. Combine digested b2AA9 and TAAR1 products so they reanneal at their AvrII sticky ends.

8. Combine new b2AA9-TAAR1 hybrid product with digested pGHE and allow reannealing at AflII (5’) and SacII (3’).