Summary: PCR mutagenesis. PCR mutagenesis is simple method for generating site-directed mutagenesis. This method can generate mutations (base substitutions, insertions, and deletions) from double-stranded plasmid without the need for subcloning into M13-based bacteriophage vectors and for ssDNA rescue. The procedure involves a PCR reaction using a supercoiled plasmid vector as the template and two synthetic oligonucleotide primers containing the desired mutation with each complementary to the opposite strands of the vector. After PCR, the template (wild type) plasmid which is dam methylated in almost all E. coli is removed by digestion with Dpn I which is specific for methylated DNA. The mutated plasmid remains intact in the reaction.
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Protocols
- Explanation and FAQs of PCR Site-Directed Mutagenesis (Stanley Maloy Lab, San Diego State University)
http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/...
Added: Mon Feb 18 2013, Hits: 1614, Reviews: 0 Write review Cached - PCR Mutagenesis (Matt Lewis, Department of Pathology, University of Liverpool)
This method uses a proof-reading polymerase to read all the way around a plasmid and thus incorporate the primer as the new (mutant) sequence. Only a few (say 12) PCR cycles are performed on a relatively large amount of plasmid template to minimise the chance of expanding PCR sequence errors.
http://www.methodbook.net/pcr/pcrmut.html
Added: Fri Sep 06 2002, Hits: 6776, Reviews: 0 Write review Cached - PCR Mutagenesis Protocol (Michael Bumbulis, Baldwin-Wallace College)
PCR is based on the enzymatic amplification of a DNA fragment that is flanked by two oligonucleotide primers that anneal (stick) to opposite strands of the target sequence. The primers are oriented with their 3' ends pointing towards each other (recall that DNA is synthesized in the 5' to 3' direction). These primers are then extended by the polymerase enzyme. Repeated cycles of heat denaturation, annealing of the primers to their complementary sequences and extension of the primers result in the amplification of the segment defined by the 5' ends of the primers...
http://homepages.bw.edu/~mbumbuli/molbio/labs/pcr/...
Added: Mon Feb 18 2013, Hits: 2603, Reviews: 0 Write review Cached -
DpnI cleavage-mediated Site Directed Mutagenesis
(Nonet Lab, Washington University in St. Louis)
A highly effective simple method for making site directed lesions in plasmids without subcloning
http://neuroscience.wustl.edu/nonetlab/ResourcesF/...
Added: Wed Feb 04 2009, Hits: 2734, Reviews: 0 Write review Cached - A Single Step Procedure to Mutagenize Multiple Sites of a Gene (Protocol Online)
A single step method to obtain multiple mutations.A modification of Quickchange site-specific mutagenesis protocol.
Added: Mon Feb 18 2013, Reviews: 0 Write review -
PCR Random Mutagenesis
(Agard Lab, UCSF)
Generate mutation on a plasmid using PCR method.
http://www.msg.ucsf.edu/agard/Protocols/PCR_Random...
Added: Wed Feb 04 2009, Hits: 1257, Reviews: 0 Write review Cached -
Site Directed Mutagenesis
(Kingsley Lab, Stanford University)
This protocol can be used for substituting individual base pairs, adding tags, making deletions, pretty much anything
http://kingsley.stanford.edu/Lab%20Protocols-WEB%2...
Added: Mon Feb 18 2013, Hits: 1389, Reviews: 0 Write review Cached -
Site-Directed Mutagenesis (Stratagene protocol)
(McManus Lab, UCSF)
This is the protocol for site-directed mutagenesis based on the Stratagene kit.
http://mcmanuslab.ucsf.edu/protocol/site-directed-...
Added: Mon Feb 18 2013, Hits: 1016, Reviews: 0 Write review -
Site-directed Mutagenesis using PCR
(Lazo Lab)
PCR site-directed methods allow site-specific mutations to be incorporated in virtually any double-stranded plasmid; eliminating the need for M13-based vectors or single-stranded rescue.
http://wheat.pw.usda.gov/~lazo/methods/lazo/pcr-mu...
Added: Tue May 14 2002, Hits: 1701, Reviews: 0 Write review Cached