New answers tagged cloning
Purification and isolation of DNA bands by cutting them from agarose gel is commonplace (Lee et al., 2012). The purification step after excision of the band gets rid of most of the EtBr and other impurities. E.g., see the websites from Isogen and Sigma-Aldrich. Reference - Lee et al. J Vis Exp (2012); 62: e3923
In this paper (Li and Elledge, Nature Genetics, 2005) it is shown that the PGK promoter works in E.coli but it has very little activity. So, I don't think it is suited to properly express resistance enzyme. About Neomycin, many aminoglycosides resistance enzymes (not all) that work on Kanamycin works well also with Neomycin. I have personally selected ...
PGK is mammalian (murine) phosphoglycerate kinase promoter. It will therefore not work in bacterial systems. Why don't you select using ampicillin only? You can increase the ampicillin concentration if you want. Regarding: I can't find anyone selecting bacteria by Neomycin anywhere. Kanamycin is generally used for bacterial selection. The Neomycin ...
In any PCR reaction ( including the assembly PCR ) the substrate of the reaction is a mix of mono nucleotides. Specific oligos serve as primer by annealing on the template and that are extended by a DNA polymerase. It is a polymerization reaction. In the LCR there are no mono nucleotides instead you have many different oligos that anneal one after the other ...
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