Could anybody suggest me what is appropriate host strain for pNZ vector?
I am using plasmid from L. Lactis and now I need to find out appropriate complement cell.
Best regards,
Stefan,
In general, DH5alpha and DH10B are good strains to try for this purpose since they lack restriction/modification systems that can prevent plasmid propagation, lack endonuclease activity that can lead to cleavage of certain plasmid sequences, and carry mutations that enable the uptake of large plasmids. Additionally, these strains are recA-, which makes them defective in homologous recombination - this avoids the potential problem of integration of your plasmid into the E. coli chromosome. These are common strains that are commercially available; the attached link is just one example.
Good luck!
http://www.mclab.com/Dh5-Alpha-Competent-E.-Coli.html
Stefan,
In general, DH5alpha and DH10B are good strains to try for this purpose since they lack restriction/modification systems that can prevent plasmid propagation, lack endonuclease activity that can lead to cleavage of certain plasmid sequences, and carry mutations that enable the uptake of large plasmids. Additionally, these strains are recA-, which makes them defective in homologous recombination - this avoids the potential problem of integration of your plasmid into the E. coli chromosome. These are common strains that are commercially available; the attached link is just one example.
Good luck!
http://www.mclab.com/Dh5-Alpha-Competent-E.-Coli.html
Dear Dante, Thank you very much for your kind reply.
I was comparing prices of 5alpha and DH10B, and more or less they are expensive. Since my grant is little bit limited. Do you think I have other options or you firmly recommend me these two strains?
In our lab we had problems expressing pNZ vector in DH5 alpha.
E. coli MC1061 can be used for cloning. check the details
http://www.bocascientific.com/docs/NICE.pdf
Normally we use DH5alpha, DH10B or TG1 for the purpose of amplification or transformation after mutagenesis or cloning to repair the nicks. As Rothangmawi mentioned they had problems expressing them in DH5alpha, we never used DH5alpha for expression. Normally, BL21 DE3 strain, XL10 Gold or BL21 Rosetta are commonly used strains for expression.
If you are out of budget for purchasing the competent cells, you may ask some of your collaborator labs or colleagues to provide you with the plates with streaked competent cells on it or a overnight culture of competent cells and then you may make your own competent cells either by Calcium chloride method or TSS method. You may alternatively make ultracompetent cells by using Tbf buffer which generally gives very high efficiency.
According to the manufacturer, pNZ plasmids, or at least some of them, are not recommended to be transformed to recA- bacteria.
Source: https://www.bocascientific.com/docs/NICE.pdf
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