I have a question regarding cloning.
I want to amplify a gene from a template plasmid, which I already have. The sequence reads -5’ TTA CDS CAT -3’
The reason the sequence is reversed is because it is under a promoter and the protein is being translated into the opposite direction.
How do I design my primers so I can get the protein sequence to read from 5’-3’ in the opposite orientation e.g. 5’- ATG CDS TAA-3’
Your gene is: 5'-ATG CDS TAA-3', So,
Forward primer: 5'-ATGxxxxxxxxxxxxx-3'
Reverse primer: 5'-TTAxxxxxxxxxxxxxx-3'
It sounds as if your plasmid is already the expression construct you need. There is no opposite direction as long as the promoter precedes the ATG start codon. Otherwise, follow Dr. Yau's recommendation. For cloning purposes you may include a restriction site suitable for your vector preceded by ~3 nucleotides (eg. 5'-ata AAGCTT ATGxxxxxxxxxxx-3'). After digestion, in this example with HindIII, you may clone your PCR product in a vector digested with HindIII and a blunt end enzyme. The HindIII site has to be 3' of the promoter.
It depends on your target vector; as suggested by the others choose appropriate restrictions sites, preceded by x nucleotides (at the homepage of New England Biolabs you can find a list with the names of the enzymes and the number of additional nucleotides to obtain full cleavage after PCR) and add the sequence of your cDNA. Because you are working with a plasmid as template it will sufficient to take 15-18 gene specific bases after the restriction site.
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