transformation of competent cells with recombinant plasmids - (Jul/14/2006 )
Hi, i would like to ask about the rationale for the possible controls 1) TE buffer 2) Uncut pUC 18/19 and 3) Digested pUC18/19 for the transformation of competent Escherichia coli DH5a with recombinant plasmids..
I assume you're trying to clone something into pUC vector by restriction and ligation?
I would never do a TE-buffer control-transformation, but the rationale might be that you could have some plasmid contamination in your buffer, which will transform and give false positive results. It might also be to check if your selective antibiotic is working (but in that case I would just transform without even adding TE to the competent cells). If you're antibiotic is still active, you shouldn't see any colonies.
Uncut plasmid transformation is to check wether your cells are still competent. You should get loads of colonies.
Digested plasmid is to check how efficient your digestion step was. If this one wasn't efficient enough, you will get a high background on your ligation plate as well, so it would be harder to find "true positives".
In case you cut with 2 different enzymes, creating different ends, you might also wanna do a control of the vector only ligation, you never know if a vector is cut with just one of the two enzymes, which would not allow ligation of your insert. Vectors cut with only one enzyme will easily 'self-ligate' and thus create high background.
As a control you need to do the self-ligation of digested vector if two enzymes were used to cut your vector. If one enzyme was used I'll suggest that after transforming your cells do the colony PCR using insert specific primers and determine the percentage of insert harboring cells.
I'm also struggling with getting good transformation efficiency with both chemical and electrocompetent cells (E. coli TG1) with recombinat plasmid.
But my problem is that I get more transformation effeciency with the control vector (10pg pUC19) but when I test the cells with my uncutted vector (20ng) the effeciency is 10 000X less than that of pUC19.
Is the decrease in effeciency due the the concentration of my vector DNA or will an increase the plasmid DNA concentration results in an increase in cell effeciency?