Batf3, Batf, and Irf4, all with N-terminal flag, were cloned into the pMYs-IRES-GFP retroviral vector. The empty vector was used as a GFP control. Mutations were cloned into the Batf3 vector using the GeneArt Site-Directed Mutagenesis PLUS kit and primers listed in
Site directed mutagenesis plus kit
Manufactured by Thermo Fisher Scientific
The Site-Directed Mutagenesis PLUS kit is a laboratory tool used to introduce specific mutations into DNA sequences. It provides a step-by-step process for efficiently modifying target genes or plasmids.
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Lab products found in correlation
2 protocols using site directed mutagenesis plus kit
1
Retroviral Transduction of Murine T Cells
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Batf3, Batf, and Irf4, all with N-terminal flag, were cloned into the pMYs-IRES-GFP retroviral vector. The empty vector was used as a GFP control. Mutations were cloned into the Batf3 vector using the GeneArt Site-Directed Mutagenesis PLUS kit and primers listed inSupplemental Table 1
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Batf3, Batf, and Irf4, all with N-terminal flag, were cloned into the pMYs-IRES-GFP retroviral vector. The empty vector was used as a GFP control. Mutations were cloned into the Batf3 vector using the GeneArt Site-Directed Mutagenesis PLUS kit and primers listed in
2
Site-Directed Mutagenesis of MDC1 Variants
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Site-directed mutagenesis on MDC1 WT and mutant plasmids (ΔFHA, ΔSDTD, ΔS/TQ, ΔPST, ΔBRCTs) was carried using ''GeneArt Site-Directed Mutagenesis PLUS kit'' (#A14604) to gain resistance against siMDC1 siRNA (5′-UCCAGUGAAUCCUUGAGGU-3′, listed in Table S2) according to manufacturer instructions. Pfx DNA polymerase was used for the amplification step due to its ability to amplify with high efficiency large DNA templates (up to 14kb) that was ideal for our experimental setup considering the size of the pcDNA3-HA-MDC1 plasmids (around 12kb). Two rounds of site-directed mutagenesis have been carried out resulting in a total number of 4 nucleotide substitutions in the coding sequence of MDC1 WT and deletion mutants with no subsequent alteration in the open reading frame (silent mutations). Two pairs of DNA oligos have been designed accordingly, carrying two altered nucleotides (point mutations) each. MDC1 mut#1 oligos: Forward: 5′-GAGCAATCCAGTGAATCGCTGAGGTGTAACGTGGAG-3′ Reverse: 5′-CTCCACGTTACACCTCAGCGATTCACTGGATTGCTC-3′ MDC1 mut#2 oligos:
The siMDC1 siRNA used in our experiments is complementary to the highlighted sequence that exists close to the 5′ end of the MDC1 coding sequence detected in both the WT and deletion mutants.
The siMDC1 siRNA used in our experiments is complementary to the highlighted sequence that exists close to the 5′ end of the MDC1 coding sequence detected in both the WT and deletion mutants.
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