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