There are two ways to add a sequence to the primer: To clone the gene you need a BspE1 site at the 5' end of the primer. Select to make a primer for the Top strand: To transform the selection into a primer expand Primers in the top menu and select Add primer. As you elongate your selection, SnapGene automatically displays the length and Tm of the selection: Go back to the Sequence view of mRFP1-Rab5 and select a sequence that starts with the ATG start codon of Rab5. The only thing that is still variable in this case is the length of the primer, which will be determined by the Tm that we wish. We want primers with a Tm in the range of 60-62☌. The forward primer should also contain a BspE1 restriction site to fuse the gene to AcGFP1. ![]() For this, our choice of primers is restricted: the forward primer needs to start with the Rab5 start codon so its location is fixed. For cloning this is often the case.įor instance, we want to make primers to clone the Rab5 gene from plasmid mRFP1-Rab5 as a fusion to the AcGFP1 gene in plasmid pAcGFP1-C1. Only when you have no flexibility in your choice of primers, I would use SnapGene to design them.
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