QuikChange Multi Site-Directed Mutagenesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- XL10-Gold cells are resistant to tetracycline and chloramphenicol. If the mutagenized plasmid contains only the tetR or camR resistance marker, an alternative strain of competent cells must be used	- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.

Upstream tips
- XL10-Gold cells are resistant to tetracycline and chloramphenicol. If the mutagenized plasmid contains only the tetR or camR resistance marker, an alternative strain of competent cells must be used
Protocol tips
- Use only the Dpn I enzyme provided; do not substitute with an enzyme from another source.

Publication protocol

Plasmid constructs
A 450-bp fragment of the uPAR promoter ( −402/+48 region) and the luciferase gene present in the pAd-TrackuPARLuc [3] were excised and cloned into a pGEM-T vector (puPARLuc). The reporter plasmid puPARLuc-NF-κBmut was generated by directed mutagenesis. The NF-kB binding site in the uPAR promoter (located at −45 bp of transcription initiation site) 5'-GGGAGGAGT-3' [13], was mutated to 5'-GGATCCAGT-3', using the QuikChange Multi Site-Directed Mutagenesis Kit (Agilent, CA USA), as recommended by the manufacturer and using the mutagenesis primer: 5'-ctctttcgcaaaacgtctggatccagtccctggggccacaaaac-3'. puPARE1A was generated by cloning the uPARE1A construct in a pGEM-T vector. The NF-κBmutation in the puPARE1A to generate puPARE1A-NF-κBmut was carried out by directed mutagenesis as described above. 3xκB-Luc reporter plasmid was previously described [31].

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Site Directed Mutagenesis (SDM) Human - Point mutation BxPC-3 uPAR using QuikChange Multi Site-Directed Mutagenesis Kit from Agilent Technologies.

Manufacturer protocol

Download the product protocol from Agilent Technologies for QuikChange Multi Site-Directed Mutagenesis Kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Site Directed Mutagenesis (SDM) Human - Point mutation BxPC-3 uPAR using QuikChange Multi Site-Directed Mutagenesis Kit from Agilent Technologies. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.