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Site directed mutagenesis
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i would like to have information about the methods of oligonucleotide ligation assay
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these questions are good ones but some suggestions ahould also be mentioned
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I get colonies on my negative control. This is the control that had the template DNA but no primers during the PCR reaction. This means that Dpn1 is not cutting the parental strand. I cannot get Dpn1 to cut the parental strand. What should I do?
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Try to rise the Ampcillin in your plates, and also in your MPs. So, the Bacteria which will grow will contain the plasmid. Be sure from your DNA, is it good? Innoculate more than the ordinary 16hr. Douple and triple in your MPs. Hope at the end you could get a result. Good Luck.
Samy
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I just experienced the identical problem like acrs(colonies, but no DNA in miniprep)
the technical support from Stratagene suggested: a) do a PCR on the colonies to find out whether they contain the original plasmid
b) streak the XL10GOLD cells on the same selective medium (contaminants?) however
so far I did not find a solution and would appreciate any comments
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Hello,
I have a bit of a problem lately doing site directed mutagenesis. I am using stratagene's kit and everything is fine up till the colony formation stage. That is I do get colonies on my plate ((between 10-50/plate). All my problem currently lies in the fact that after pikcing colonies and doing miniprep, I don't get so see any plasmid there. This has been very very consistent. Many colonies picked but non having plasmid. Ironically, a control puc plasmid do show on agarose.
Would greatly appreciate any input,
MANY THANKS
Acrs
Rating: Good
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Hello,
I have a bit of a problem lately doing site directed mutagenesis. I am using stratagene's kit and everything is fine up till the colony formation stage. That is I do get colonies on my plate ((between 10-50/plate). All my problem currently lies in the fact that after pikcing colonies and doing miniprep, I don't get so see any plasmid there. This has been very very consistent. Many colonies picked but non having plasmid. Ironically, a control puc plasmid do show on agarose.
Would greatly appreciate any input,
MANY THANKS
Acrs
Rating: Good
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You can do the site directed mutagenesis by cloning the fragment of the gene of interest in a suicide vector and then introducing it into wild type strain through homologous recombinations via conjugation or electroporation
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i would like to have information about three methods of site directed mutagenesis
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Related resource
Site-directed mutagenesis
Internal deletions by PCR
Mutagenesis for deletions
Dut/ung in vitro Site-directed Mutagenesis Protocol
Some commercial kits and their brief introduction (biocompare)
Site-directed Mutagenesis using PCR
QuikChange(tm) Site-Directed Mutagenesis Kit
DpnI mediated site-directed Mutagenesis
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Related new technical papers
Attempting to remove the substrate inhibition of L-lactate dehydrogenase from Bacillus stearothermophilus by site-directed mutagenesis. Appl Biochem Biotechnol. 2007 May-Jun;141(2-3):265-72
One-step site-directed mutagenesis of viroid dimeric cDNA. J Virol Methods. 2007 Oct;145(1):71-5
Chain length-dependent binding of fatty acid anions to human serum albumin studied by site-directed mutagenesis. J Mol Biol. 2006 Oct 27;363(3):702-12
A site-directed mutagenesis method utilising large double-stranded DNA templates for the simultaneous introduction of multiple changes and sequential multiple rounds of mutation: Application to the study of whole viral genomes. J Virol Methods. 2006 Oct;137(1):63-71
Automated high-throughput process for site-directed mutagenesis, production, purification, and kinetic characterization of enzymes. Anal Biochem. 2006 Aug 1;355(1):110-6
SiteFind: a software tool for introducing a restriction site as a marker for successful site-directed mutagenesis. BMC Mol Biol. 2005;6:22
Sulfhydryl groups responsible for extreme lability of human cholesterol 7alpha-hydroxylase identified by site-directed mutagenesis. J Steroid Biochem Mol Biol. 2003 Jul;86(1):35-40
Development of antibiotic-overproducing strains by site-directed mutagenesis of the rpsL gene in Streptomyces lividans. Appl Environ Microbiol. 2003 Jul;69(7):4256-9
Cloning of the genomes of human cytomegalovirus strains Toledo, TownevarRIT3, and Towne long as BACs and site-directed mutagenesis using a PCR-based technique. Virology. 2003 Mar 1;307(1):164-77
[Site-directed mutagenesis of atrial natriuretic peptide gene and effect of the mutations on its diuretic activity in nephrotic rats] Zhonghua Yi Xue Za Zhi. 2002 Oct 10;82(19):1324-7
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