Quikchange 2 pcr protocol

Full-length cDNA for KDM5A (JARID1A, P29375), KDM5B (JARID1B, Q9UGL1, cDNA), KDM5C (JARID1C, P41229, IMAGE 5492114), KDM4A (O75164) containing A482E substitution (natural variant rs586339), KDM4B (JMJD2B, O94953, gift from ICR), KDM4C (JMJD2C, Q9H3R0, 8143862), KDM3A (JMJD1A, Q9Y4C1, 4823253), KDM2A (FBXL11, Q9Y2K7, 5534384) and KDM6B (JMJD3, O15054, cDNA) were amplified by PCR from either a MGC clone or commercial cDNA source or received as a gift from collaborators and cloned into pDONR-221 vector using Gateway BP reaction producing Gateway entry clones.
To produce catalytically inactive KDMs residues involved in iron coordination were substituted for alanine (KDM5A H483A/E485A, KDM5B H499A/E501A, KDM5C H513A/E515A, KDM5D H510A/E512A, KDM4A H188A/E190A, KDM4B H189A/E191A, KDM4C H190A/E192A, KDM3A H1120Y, KDM2A H212A/D214A and KDM6B H1390A/E1392A). Mutations were introduced into full-length KDM Gateway entry clones using 15 cycles QuikChange II PCR protocol (Agilent Technologies).
Mammalian expression constructs encoding for N-terminal 3*FLAG potencies were constructed using the Gateway LR recombination reaction between pCDNA5-FRT/TO-3FLAG destination vector [40 (link)] and the wild-type or mutated KDM Gateway entry clone.
All constructs described are available upon request.

The BRD4/ET mutant (BRD4/ET^mut, D650K/E651K/E653K/D655K) was cloned using a two-step PCR. First, the C-terminal part was amplified using a long forward primer bearing the mutations and the BRD4/ET reverse primer. The PCR product was then purified from an agarose gel and used as a degenerated primer during the amplification of the full-length PCR fragment, in combination with the BRD4/ET forward primer. Mutations which impede binding to a first (N116F) or second (N391F) bromodomains were introduced into the full-length BRD3 Gateway entry clone using a 15-cycle QuikChange II PCR protocol (Agilent). Mutations or deletions which impede binding to the BRD4 ET domain were introduced into full-length BRD9 (Uniprot: Q9H8M2; BRD9^mut: K29A/V31A/K33A; BRD9^del: deletion of D18-G36); WHSC1L1 (Uniprot: Q9BZ95; WHSC1L1^mut: K154A/L155A/K156A; WHSC1L1^del: deletion of V143-I161); ZNF592 (Uniprot: Q92610; ZNF592^mut: K374A/V375A/R376A; ZNF592^del: deletion of V364-T379) in Gateway entry clones using the same QuikChange II protocol.