pGEM methylation status - (May/17/2007 )
does anybody have an idea of the methylation status of the pGEM vector after it is transformed into competent cells?
Reason why I ask: would it be possible to grow up competent cells with the vector, extract plasmid DNA, and treat with/without sss1 to get positive and negative control/ standard curve?
that would depend on your strain of bacteria that you are transforming,
we use a DH5 alpha which is methylation negative and the plasmids you isolate are unmethylated, we then SssI methylate our constructs.
DH5alpha seems to be positive for DAM and DCM genes (http://www.promega.com/enotes/faqspeak/0011/fq0026.htm)
JM110 is dam dcm minus :
JM110 Genotype: rpsL (Str) thr leu thi-1 lacY galK galT ara tonA tsx dam dcm supE44 (lac-proAB) [F´ traD36 proAB lacI Z M15].
genes on F' episome are wild type
so it is fred33 then why are our cells labelled DH5-alpha!!!
the plasmids we isolate from the bacteria are unmethylated by hpaII digestion.....hmmmmmmmmm
errrrrrrrrrrr then i don't know what to say to bram except check it ...
dam and dcm will not specifically target CG positions. dam methylates the A in GATC seequences, while dcm methylates the second C in CCWGG sequences. Neither will have any effect on HpaII sequences. The sssI assay should work well with plasmids from virtually any E. coli strain. In very obscure cases, you could see methylation and cutting from the EcoKI hsd system.
The main effect of bacterial methylation in working with eukaryotic DNA is the presence of the mcrA gene, which can cut at methylated CG sites. This can be a problem if you are attempting to clone DNA from a eukaryotic source directly into bacteria (without, e.g., doing a PCR reaction on it).
thanks for clarifying that up phage434.
Was getting a little worried with what we were transforming!