I am cloning a gene into a vector that has the reporter gene LacZ, but I am getting some background and almost no clones. If I use another enzyme, to cut the reporter gene LacZ, would it be a good choice to reduce background? Thanks a lot
Hi there,
What do you mean by background as you say you are not getting a lot of clones? If you get only few clones, it means that your ligation and/or transformation is not efficient enough. The ligation efficiency depends on the quality of your linearized vector and insert (in terms of compatible ends and in terms of molar ratio used between the two for ligation). If you are using one single restriction enzyme, linearization of the vector is easily spotted on an agarose gel whereas the digest of the DNA insert is not (in case your insert is generated by PCR) therefore don't hesitate to digest it for a longer time. Keep in mind that some restriction enzyme are affected by the proximity between the restriction site and the end of the PCR product... I usually gel-purify vector and insert after digest and quantify DNA with a Nanodrop. About ligation, you have to use 100ng or less of the vector and the suitable amount of insert to respect a 1:3 to 1:5 molar ratio. I systematically include two controls : one without ligase and one without insert. They will give you a rough idea about the non digested vector present in your vector prep and about the frequency of self-ligation event, respectively. The very last control I perform is to test transformation efficiency of your recipient strain using 0.1ng of circular plasmid (usually pUC18). If you get less than 106 clones per µg, your cells are not suitable for transformation with ligation mix.
The choice of an other enzyme will be judicious in case your problem is linked to digestion efficiency...
Hope this will help...
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