I am trying to clone gene of my interest along with tag to pET24 d (+) vector. As the insert has its own ATG, at the start and another ATG in other frame which is showing restriction site for No 1, so if i design a restriction primer to isolate CDS from whole sequence it will ultimately truncate the CDS, so please guide me what strategy should i use to complete isolation and expression of CDS.
I suggest you to perform two constructs: the first with the whole sequence and the second starting with the second atg.
My initial suggestion would be to see if the internal NcoI site can be removed by introducing a silent mutation using in vitro mutagenesis. If that is possible, then you will be able to excise your silently mutated CDS from your cloning vector and ligate it into the pET vector.
Change vector with an NdeI restriction site unsteast of NcoI. Or Make a fusion protein with anotther sites or mutagenesis.
Two possibilities :
1) If NdeI restriction site is absent from your CDS, try to use this site for cloning your CDS into pET24a for instance (ie design a primer creating NdeI restriction site at 5', "transforming" NcoI site into NdeI site. This should not modify your coding sequence as introducing a NdeI consensus sequence CATATG doesn't affect sequence downstream the ATG...)
2) just run cloning experiment as if there was not second NcoI site in the CDS. As a result NcoI digest of your PCR product will generate two fragments because of the presence of the second NcoI site. Instead of gel purifying perform DNA extraction and precipitation. Then proceed to ligation using the precipitate as the insert sample. It should contain stoechiometric amounts of the two compatible fragments you want to clone. Theoritically, half of the clones you will obtain should be right relative to the NcoI-NcoI fragment orientation towards the downstream fragment.
Hope it will help...
In case you got some dollars to spare, Gibson assembly is best. no tranditional restriction involved as it uses homologous recombination. The process involves designing primers which incorporate 20-30bp homology (to the cloning site sequence) upstream and downstream your gene of interest. I been told In-fusion cloning works similarly. These are possible options in any case you wanna completely avoid restriction. Gibson kit is about 120 gbp in UK (10 reactions)
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