Team:Hong Kong HKU/methodsnprotocols

PCR: PCR was performed with either (1) GoTaq Flexi G2 Polymerase from Promega, or (2) LongAmp polymerase from NEB (if the PCR product is >2000bp), or ExTaq polymerase from TaKaRa.
DNA purification: Nucleic acid extraction and purification from gel or reaction mixtures was all performed with QIAquick gel extraction kit and QIAquick PCR purification kit.
Restriction: Restriction enzymes are from NEB with their suggested buffers and double digest guidelines given online. Ligation: T4 DNA Ligase from NEB or Invitrogen (ExpressLink T4 DNA Ligase and H.C. T4 DNA Ligase) was employed, ligation times and temperatures varies as specified by the suppliers.
TA cloning: TA cloning was carried out for some PCR products by non-proofreading polymerases; PCR products amplified by LongAmp polymerase has its 3'-A added by incubating with GoTaq Flexi polymerase from NEB
Transformation: Ligation mix were always transformed into competent DH10β cells using the heat-shock method; constructed plasmids for expression were transformed into competent BL21(DE3) cells using electroporation. Recovered cells were plated on LB plates supplemented with the appropriate antibiotics. Both strains were purchased from NEB and expanded and made competent ourselves.