IPCR

INVERTED PCR/BIOBRICK CLONING

1. Conduct regular PCR of insert/part B (eg. AdhE from J100107) using Prefix_For and Suffix_Rev primers 2. Purify PCR product and digest with XbaI + PstI 3. Heat inactivate restriction enzymes at 80 C for 15 minutes 4. Conduct Inverted PCR of vector with part A (eg. promoter + C.dog in S05022) using Spe_Rev and Pst_For primers 5. Purify PCR product and digest with SpeI + PstI 6. Heat inactivate restriction enzymes at 80 C for 15 minutes 7. Ligate the two PCR products and transform bacteria

Prefix GAATTCGCGGCCGCTTCTAGAG CTTAAGCGCCGGCGAAGATCTC

Suffix TACTAGTAGCGGCCGCTGCAG ATGATCATCGCCGGCGACGTC

Prefix_For; TGCATGAATTCGCGG

Suffix_Rev; TGCATCTGCAGCGGC

Spe_Rev; GCATCCGCTACTAGTA

Pst_For; TGCATGCCGCTGCAG