I am trying to clone a gene that has a NdeI site in the sequence, so I wouldn't be able to use NdeI site in the forward primer for start codon. Apart from using NcoI, what other options are available? Since I have cloned similar genes using NdeI/XhoI in pET21a and would prefer to clone the new gene too in the same vector. Will cloning the gene with ATG after the BamHI/EcoRI site result in a protein with extra amino acids at the N-terminal? Alternatively, is there an expression vector where I can clone the gene without using NdeI or Nco I and without any tags?
Hi there,
In bacterial expression plasmid it is important to use the initiation codon placed at the right distance fro RBS sequence (in pET21 it is the ATG in NdeI site). So if you want to clone between BamHI and EcoRI, you will have to respect the reading frame imposed with the ATG in the NdeI site. Therefore the translation product will contain a few aa on the Nter derived from the sequence between NdeI and BamHI. If you don't want any tag and limit the number of extra aa on the Nter you should use pET 21 and use NheI site as the upstream cutting site (tripeptide met-ala-ser will be at Nter) and any downstream site. When designing the reverse primer you will have to include a stop codon immediately upstream the cutting site so that it will interrupt translation without reading downstream tag.
My favorite cloning method for everything except AAV is to use CPEC (circular polymerase extension cloning, similar to Gibson, but cheap) which is ligation and sequence independent. That way you don't add anything extra and it doesn't matter that you don't have all your favorite cut sites.
Basically, you design primers at the borders of your insert (insert-F and insert-R), and another set of primers at the borders of your vector (vector-F and vector-R). Then you add the vector primer to the tail of your insert primers and your insert primers to the tail of your vector primer such that there would be significant overlapping region. Basically the two sets of primers should be reverse complements of each other: Insert-F should be reverse compliment of vector-R and insert-R should be reverse complement of vector-F.
Now you run two separate PCRs, one for the insert and one for the vector. Then do a DpnI digest to get rid of the template. You can also do a PCR cleanup if you want to get rid of the original primers, but it's not necessary. Now mix the two products together and do a second PCR. The insert will now act as a primer for your vector and vice versa, creating a complete plasmid. The plasmid will be nicked in two places. It's not necessary to ligate this because when you transform it, the bacterial DNA repair machinery will fix it.
I use this technique all the time and I have 75-90% success rate on my most difficult clonings. I usually only pick 1 or 2 colonies because it's so robust.
I included a diagram modified from: (Figure 4) Chapter Imaging Glutamate with Genetically Encoded Fluorescent Sensors
I prefer the TOPO cloning kit. As long as your PCR product has an A overhang, you can directly insert into the TOPO plasmid. It's a bit expensive, but worth the cost because it is very effective.
https://www.thermofisher.com/us/en/home/life-science/cloning/topo.html
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