High fidelity pfu ultra 2 polymerase

A fully functional mouse MR fluorescent derivative with insertion of enhanced green fluorescent protein (eGFP) after amino acid 147 has been previously described (Aguilar‐Sanchez et al., 2012 (link)). N‐terminus eGFP tagged wild type mouse GR or truncated mutant GR‐N525, lacking the entire LBD, (eGFP‐GR and eGFP‐GRN525, respectively) have been previously described (Meijsing et al., 2007 (link); Presman et al., 2016 (link)). A plasmid expressing mouse GR tagged in the N‐terminus with mCherry was developed by amplifying mouse GR coding sequence and in‐frame cloning in pmCherry‐C3 (Clontech). Point mutations and deletions were introduced using the Quickchange XL mutagenesis kit (Agilent) following the manufacturer's instructions. Domain swapped mouse MR/GR constructs were constructed by amplification of the relevant fragments from donor plasmids and directional cloning using ligation‐free In‐Fusion technology (Clontech) in PCR‐mediated linearized vectors. PCRs were performed using high‐fidelity Pfu ultra II polymerase (Agilent). All constructs and mutations were confirmed by DNA sequencing.

A fully functional mouse MR fluorescent derivative with insertion of enhanced green fluorescent protein (eGFP) after amino acid 147 has been previously described (Aguilar-Sanchez et al., 2012 (link)). N-terminus eGFP tagged wild type mouse GR or truncated mutant GR-N525, lacking the entire LBD, (eGFP-GR and eGFP-GRN525, respectively) have been previously described (Meijsing et al., 2007 (link); Presman et al., 2016 (link)). A plasmid expressing mouse GR tagged in the N-terminus with mCherry was developed by amplifying mouse GR coding sequence and in-frame cloning in pmCherry-C3 (Clontech). Point mutations and deletions were introduced using the Quickchange XL mutagenesis kit (Agilent) following the manufactureŕs instructions. Domain swapped mouse MR/GR constructs were constructed by amplification of the relevant fragments from donor plasmids and directional cloning using ligation-free In-Fusion technology (Clontech) in PCR-mediated linearized vectors. PCRs were performed using highfidelity Pfu ultra II polymerase (Agilent). All constructs and mutations were confirmed by DNA sequencing.