Notice that the black arrows after the Ns? Those are the actual cleavage sites. If you look at the diagram for BspMI, you'll see that the top strand has N4 at the 3' end on the top strand and N8 at the 5' end on the bottom strand. Methylation-specific nucleotides show a CH3 on NEB's website, like for DpnI, so no methylation is not necessary. The Ns mean any nucleotide, the subscript refers to the numbers of nucleotides. You say BspMI, but your link is to BpmI instead. THANK YOU! (Sorry for the potentially dumb questions No one else in my lab in a evolutionary bio lab does molecular work and cloning) I am very confused about how I should design the forward primer in the case of BspMI, in terms of how many bp should flank the cut site upstream and downstream in the primer design, what exactly is the cut site (documentation says: CTGGACn16 do I need to methylate my DNA before cleavage?)ĭoes anyone have experience using/designing primers for inserting BspMI cut sites? Could anyone pretty please give an example of what a correctly designed primer for inserting this cut site should look like? Unfortunately, the gene I want to clone into an expression vector (pET20) has MANY restriction cut sites, and BspMI (forward) and NotI (reverse) are the only combo that won't cut my gene: I am a newb to cloning with restriction enzymes that I haven't used before, and I need to introduce BspMI as a cut site in the forward direction of a gene that I am trying to clone:
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