Site directed mutagenesis - wild type clone (Apr/21/2006 )
Hello all,
I am new to this field and would hope to get some help about site directed mutagenesis. The gene fragment of interest is 1.5kb, and I would like to create a mutant site in this gene. I am not sure if the following is correct - Before doing site directed mutagenesis, am I supposed to do a pcr of the gene of interest, clone it into a vector and do a sequencing to make sure that it is wild type? And does the gene of interest has to been 100% wild type in the WHOLE 1.5kb gene (100% match to the Pubmed published sequence?)?
Looking forward to your hearing your comment/advice.
Seong
I think so
what do you mean, 'the WHOLE gene'?
are you not intending to have the mutated gene on a plasmid for some sort of expression down the road? if you just need a small mutation, perhaps you could take a fragment of the gene, use PCR or SDM to get a mutant, and allow a little homologous recombination to get your mutant strain?
are you working in prokaryotes or eukaryotes? could you please give more details about what you want to do?
what do you mean, 'the WHOLE gene'?
are you not intending to have the mutated gene on a plasmid for some sort of expression down the road? if you just need a small mutation, perhaps you could take a fragment of the gene, use PCR or SDM to get a mutant, and allow a little homologous recombination to get your mutant strain?
are you working in prokaryotes or eukaryotes? could you please give more details about what you want to do?
Hello.
I need to get a clone with a point mutation related to MELAS disease, and the plasmid carrying the mutation will be used as template for PCR to amplify the 1.5kb mutated gene fragment. This gene fragment will only be used as standards for the assays of screening for DNA mutation and will not be used for protein expression. What I am thinking now is to first get a wild type clone, and use this wild type plasmid as template for the site directed mutagenesis to create a point mutation. However, I am not sure if this wild type DNA needs to be 100% matched to the published sequence in PUBMED or if it requires only the site of interest (where I will create point mutation) to be 'wild-type'.
Eventually what I need is a gene fragment (PCR product) which carries one specific point mutation to be used as standard. I am working with eukaryotic DNA.
What do you exactly mean by "the assay of screening for DNA mutation"?
Will you use it as a control for a reaction with probes for a specific part of your DNA? If so, I wouldn't be bothered with mutations outside of your probe region.
Will you use it as a control for a reaction with probes for a specific part of your DNA? If so, I wouldn't be bothered with mutations outside of your probe region.
Yes. Thank you very much.