All in one pcr cloning kit

The site on the plasmid with the random insertion was confirmed by RESDA-PCR^{43 (link)}. The PCR conditions were as described in the Kong and Li-Beisson⁴⁴ . The 1st PCR was amplified in a 20 µL volume using Taq DNA polymerase (Biofact, Korea) with degenerate primers and pEtt-F1 primers. The 2nd PCR was carried out in a 20 µL volume using Platinum II Taq Hot-Start DNA polymerase (Invitrogen, USA). The Q0 and pEtt-F2 primers were used for amplification in the 2nd PCR. The final PCR product was separated on a 1% agarose gel at 100 V for 30 min using electrophoresis. The amplified band was extracted by Wizard SV gel and a PCR clean-up system (Promega, USA). PCR fragments were cloned using the All in One PCR Cloning Kit (Biofact, Korea), cultured in LB medium with kanamycin and ampicillin (final conc. 50 µg mL⁻¹), and sequenced with M13 universal primers.

pBABE‐YAP1 plasmid DNA (15682) was purchased from Addgene (Cambridge, MA). The YAP S127A mutant construct (serine to alanine at residue 127) was generated by the introduction of a single point mutation to pBABE‐YAP1 using QuikChange Site‐Directed PCR Mutagenesis Kit (Stratagene) and verified by sequencing. To exchange the backbone vector from retrovirus‐based to lentivirus‐based, insert PCR was performed with retroviral pBABE‐YAP1 or YAP S127A plasmid as the template. TA cloning was subsequently performed using All in One™ PCR Cloning Kit (Biofact, Daejun, Korea) according to the manufacture's protocol. The multiple independent clones were isolated and confirmed by DNA sequencing. The correct plasmids were subcloned into lentiviral pLVX vector (Takara Bio Inc, Shimogyō‐ku, Kyoto, Japan, cat #632164). The direction of the ligated fragment was confirmed by restriction mapping using EcoR I and Xba I enzymes.