I have a cloning vector (fig. a) with target gene bMPRO. I want to insert this target gene into the attached vector (fig. b) for using for cell transfection.
How can I solve this problem? any easier alternative or suggestion to use this target gene for cell transfection?
Dear Islam,
i faced this problem before, as your target vector just containing EcoRI and KpnI , and this is the only possibility to clone your target gene in this expression vector, you can not use the a direct digestion method using mixture of EcoRI and KpnI, as this can not get your insert because both of them in the same flanking side, so if your genes is not long enough , i recommend to use high fidelity PCR with two flanking primers to pick your target gene from the cloning vector and then digest the PCR product and then ligated to the linearized expression plasmid.
so you have at least four stategy:-
1- flanking primers with forward EcoRI and reverse KpnI site and then go for PCR, i recommend Primestar GXL of Phusion as it is high fidelity ones, then run few microlitter from the PCR product , then check you insert size, if it is correct, then digest the remaining with mixture of EcoRI and KpnI , but you have to know both of them seems to work under different buffering condition, (high for EcoRI, and low for KpnI), but i think you can use low buffer as general with EcoRI and KpnI mixture, accroding to your compnay you purchased the enzyme from (takahara,nippon gene, NEB, or so on..) or use universal buffer, but i think EcoRI can work under different buffer condition. after that cut the expression vector with both mixture(EcoRI-KpnI) and run 1ul to check the linearziation on agarose gel. then start ligation to and tranformation,
2- if you are worry about KpnI enzyme, use F and reverse primer with EcoRI restriction site to pick up your gene , then digest your PCR product, and linearize your plasmid with EcoRI then dephosphorlated using shrimp alkaline phosphatase, to prevent self annealing, then ligate you insert and check the orientation.
3- i do not know if you have another cloning vector then like PGEM sigma T easy vector, or other that have several cloning site, so you can cut if possible you insert and ligate in shuttle vector with proper orientation, and if that shuttle vector has EcorI and KpnI in proper orientation you can digest and ligate to you target expression vector.
4- you can use the third strategy if you have problem in the cloning site, you can modify the EcorI restriction site to other site by adding linker then ligate you insert depending on the linker you will use. please let me if you want to use the last one, i can explain much more details.
warm regard`s
Sherif
Please Islam check if your insert does not contain restriction site for either ecorI or kpnI for sure or you need then to modify the cloning site in your plasmid , Sorry I firget to mention it clearly .
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