Colonies were used to inoculate overnight cultures.
Plasmids were isolated using a miniprep kit.
Amplification of 19 and 15 with Q5 High-Fidelity DNA Polymerase out of E.coli
19 amplification at 65.0°C with 19.rev/fwd primes
PCR worked, positive control worked, no amplification of 19
15 amplification at 65.0°C with 15.rev/fwd primes
PCR worked, positive control worked, no amplification of 15
A product length of 900 bp was expected. The gel shows that most plasmids seem to be correct.
This result was confirmed by sequencing.
The fragments of 19 and 15 were purified with PCR Purification Kit.
Ligation of 15 with 19
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.
Notice: This page is currently under construction. Contents in this page are temporaory and will be modified several times before the final release. — 2016 iGEM Team Tianjin
