GeneArt™ Site-Directed Mutagenesis System

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- Create a fresh dilution of 25X SAM from the kit-supplied 200X SAM in sterile, distilled water each time you perform the mutagenesis procedure - 25X SAM is not stable, and loses activity within a few hours after preparation

Protocol tips
- Create a fresh dilution of 25X SAM from the kit-supplied 200X SAM in sterile, distilled water each time you perform the mutagenesis procedure - 25X SAM is not stable, and loses activity within a few hours after preparation

Publication protocol

Plasmid constructs and siRNA transfections
Plasmids used were pCI empty vector, pCI-cIAP1, pCI-cIAP1-H588A, pCMV-βGal, pCMV-HA-DP1, pGL3 human CCNE promoter;12 pcDNA3.1-6His-Ub wt and 6His-Ub-K63-only mutants in which all K have been mutated into R except the K63; pCMV-HA-E2F1 wt and pCMV-HA-E2F1 K117/120/125R,17 pCMV-3HA-E2F1 and pCMV-3HA-E2F1-K185R;25 pCMV-Flag-E2F1 wt and mutants that contain no lysine (K0) or only K89 (K89 only), K89 and K137 (K89, 137), K89, K137, K161 and K164 (K89, 137, 161, 164) or in which K117/120/125) (cluster 1), K182/183/185 (cluster 2) and/or K226/287/289/310 (cluster 3) have been mutated into R.26 The pCI-cIAP1-F616A mutant was obtained by site-directed mutagenesis from pCI-cIAP1 with GENEART Site-Directed Mutagenesis system (Invitrogen, ThermoFisher Scientific, Villebon-sur-Yvette, France) by using the following primers: forward (5′-CAAGGGTACTGTTCGTACAGCTCTCTCATAATCGACCCG-3′), reverse (5′-CGGGTCGATTATGAGAGAGCTGTACGAACAGTACCCTTG-3′). The pCMV-3HA-E2F1 mutants (K89R, K137R, K161/164R, K181/183R, K266R, K287/289R, K310R) were obtained by site-directed mutagenesis from pCMV-3HA-E2F1 (GenScript, Piscataway, NJ, USA). Plasmids constructs were transiently transfected using JetPEI (Polyplus transfection, Ozyme, Montigny-le-Bretonneux, France). Lipofectamine RNAimax reagent (ThermoFisher Scientific, Villebon-sur-Yvette, France) was used to transfect siRNAs. RNA oligonucleotides are designed and purchased from Qiagen (Qiagen France SAS, Courtaboeuf, France) for cIAP1, Rb, cyclin A and control siRNA or Ambion (Thermo Fisher Scientific) for PRMT-1 and PRMT-5 siRNA.

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Discussion

Papers

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Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for GeneArt™ Site-Directed Mutagenesis System below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Site Directed Mutagenesis (SDM) Human - Point mutation HeLa CCNE using GeneArt™ Site-Directed Mutagenesis System from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.