Many restriction enzymes do not cut DNA efficiently at the end of a linear piece (see NEB for more information). Therefore, our Forward Primer will use the sequence 5'-ATGTGGCATATCTCGAAGTAC-3' for the region that binds the ORF and we will add the EcoRI restriction site (GAATTC) to the 5’ end of this primer, making our Forward Primer 5'-GAATTCATGTGGCATATCTCGAAGTAC-3'. Assuming you are amplifying from plasmid DNA (rather than from genomic DNA or a cDNA library), roughly 18-21bp is usually sufficient to give specificity and to also be compatible with a standard PCR reaction. The following image shows the ends of the ORF and how these are used for primer design:īecause we are cloning an ORF, we want to clone from the start codon (ATG) to the stop codon (TGA, in this example).
Next, we need to examine the DNA sequence that we want to amplify and design primers that will bind to and replicate it. Remember to insert your DNA in the correct orientation in the recipient plasmid by viewing the MCS and fusing the upstream restriction site to the forward primer and the downstream restriction site to the reverse primer. In our example, we will use EcoRI and NotI to ligate our cDNA into the recipient plasmid. You want to choose enzymes that:Īre in the desired location in your recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere on the plasmidīonus: It is helpful to choose restriction enzymes that can both function in the same buffer, as this will save time later When selecting restriction sites, you should use a DNA analysis tool, such as Addgene’s Sequence Analyzer, to allow you to identify which restriction sites are present in a given sequence. Hybridization Sequence: The region of the primer that binds to the sequence to be amplified (usually 18-21bp) Restriction Site: Your chosen restriction site for cloning (usually 6-8bp) Leader Sequence: Extra base pairs on the 5' end of the primer assist with restriction enzyme digestion (usually 3-6bp) The basic PCR primers for molecular cloning consist of:
0 kommentar(er)
