We add a single 3`-adenine overhang to each end of the fragment of 19 and then purified with DNA Purification Kit.

19 was ligated into T vectors via TA clone and transformed into E.coli via heat shock.

A colony PCR was performed with five colonies.

Gel electrophoresis showed that 5 colonies were positive for insertion of 19. Two of these colonies containing 19 were used to inoculate overnight cultures.

15 gene fragment was phosphorylated.

Plasmids containing T vector with 19(pT-19)were isolated using a miniprep kit.

Mono-restriction digest of pT-19 with stu I.

The enzyme-digested product was dephosphorylation.

Dephosphorylated plasmid and phosphorylated gene 15 were connected.

Ligation product was transformed into E.coli via heat shock.

A colony PCR was performed with twelve colonies.

Gel electrophoresis showed that 2 colonies were positive for connecting the plasmid and gene fragment. These two colonies were used to inoculate overnight cultures.

Plasmids with correct sequence of 19-15 were isolated using a miniprep kit.

Mono-restriction digest of pT-19-15 with Nru I.

The enzyme-digested product was dephosphorylation.

13 amplification at 65.0°C with 13.rev/fwd primes

PCR worked, positive control worked, no amplification of 13

The fragments of 13 were purified with PCR Purification Kit.

13 gene fragment was phosphorylated.

Ni inducible promoter was ligated into pCPC-3301 vector.

Phosphorylated 13 was ligated into pT-19-15 and transformed into E.coli via heat shock.

Single colonies were obtained by plating.

A colony PCR of Ni inducible promoter(pCPC-3301-Ni) was performed with 12 colonies.

Two of the successful ones were used to inoculate overnight cultures.

A colony PCR of pT-13-19-15 was performed with 7 colonies.

Two of the successful ones were used to inoculate overnight cultures.

Two kinds of plasmids were isolated using a miniprep kit.