The plasmid pFA6a-GFP(S65T)-His3MX6 (Longtine et al., 1998 (link)) was a gift from John Pringle (Addgene 41598; Addgene, Watertown, MA, USA) and pFA6a-link-yoTagRFP-T-Kan (Lee et al., 2013 (link)) was a gift from Wendell Lim & Kurt Thorn (Addgene 44906); both were given in the form of bacterial stabs. Note that pFA6a-link-yoTagRFP-T-Kan is the source of the KanR marker used to delete HTB2-HTA2. Five mL of bacteria were cultured overnight, then plasmids were extracted with the QIAprep Spin Miniprep Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions.
Extracted plasmids were linearized by double digestion with a reaction containing 1 µg of plasmid DNA, 5 µL of 10× rCutSmart Buffer (New England BioLabs, Ipswich, MA, USA), 10 units each of SalI and EcoRV restriction enzymes (New England BioLabs) topped up to 50 µL with nuclease-free water. The reaction mixture was heated at 37°C for 15 min for the digestion reaction, then 80°C for 20 min to inactivate the enzymes.
Extracted plasmids were linearized by double digestion with a reaction containing 1 µg of plasmid DNA, 5 µL of 10× rCutSmart Buffer (New England BioLabs, Ipswich, MA, USA), 10 units each of SalI and EcoRV restriction enzymes (New England BioLabs) topped up to 50 µL with nuclease-free water. The reaction mixture was heated at 37°C for 15 min for the digestion reaction, then 80°C for 20 min to inactivate the enzymes.
Free full text: Click here
