Fragment of 19 was phosphorylated 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.
Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.
The sequencing results for both of them were error.
Colonies containing Ni inducible promoter were used to inoculate overnight cultures.
PCR was performed to check if the gene fragments were ligated correctly.
13_ fwd and 15_rev on pT-13-19-15
Gel electrophoresis showed that it failed.
Plasmids pCPC-3031-Ni were isolated using a miniprep kit.
Several PCRs were performed to check if the gene fragments were ligated correctly.
1.13_fwd and 13_rev on pT-13-19-15
2.19_fwd and 19_rev on pT-13-19-15
3.15_fwd and 15_rev on pT-13-19-15
4.13_fwd and 19_rev on pT-13-19-15
5.19_fwd and 15_rev on pT-13-19-15
6.13_ wd and 15_rev on pT-13-19-15 The fourth and sixth ones were not successful.
Repetition: Phosphorylated 13 was ligated into pT-19-15 and transformed into E.coli via heat shock.
Repetition: Several PCRs were performed to check if the gene fragments were ligated correctly.
1.13_fwd and 13_rev on pT-13-19-15
2.13_fwd and 19_rev on pT-13-19-15 The second one was failed.
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