TOPO cloning problem - (Oct/08/2010 )
I am new in molecular biology but I have an urgent task to clone a gene which has to be expressed in a bacteria that does not contain that gene, call that "abc" negative. From this transformant, I will clone plasmid into the bacteria and use it in cell culture experiments to see if they will invade CaCo2 cells as the "abc" positive bacteria. I have done PCR and amplified the DNA from a template. The next step is to ensure that the DNA does not contain any mutations, so I have decded to clone it into a TOPO TA cloning vector for blue/white selection in order to reproduce plasmid for subsequent sequencing as adviced by a colleague. Is it waste of time to do this? What is the usefulness of using TOPO TA cloning step? Wouldn't it be quicker if I subclone the gene into the expression vector pET which I will be using to express the gene for subsequent transformation and to by-pass this TOPO cloning step. Any advice will be helpful.
I think if your PCR works fine with your gene of intrest, Then its of no use to go to this step and directly you can go to pET cloning for expression.
and select the abc+ bactetria after Lactose induction than IPTG. As it will give bacteria a kind of another stress. ( One should use or i will recomend to use IPTG for high protein yield) thatīs not necessary for this type of functional assays .
Your colleague is most likely suggesting you clone into the TOPO vector because it is very easy and quick ligation. Your chances of success with TOPO cloning are higher than traditional restriction digestion ligation. However, I would think it would be worth trying both at the same time. If the pET ligation works right away, you are lucky and can stop the TOPO approach but if the pET ligation fails, you may have success with the TOPO and can move ahead. Does your PCR primers have appropriate digestion sites that will allow you to ligate into the pET vector? If not, then you really have no choice but to go with the TOPO and then restriction digest out of the TOPO and ligate into pET. The only down side to TOPO cloning is that you must screen your insert for the correct orientation.
Which Topo TA vector is it? Many of the topo TA vectors are eukaryotic expression anyway.
First you need to check if your restriction site is difficult to cleave at the end of PCR fragment or not. Some restriction site is known to have end cleavage problem. If you can't cut it well, you can't ligate well into your pET vector.
IMO, I would do it right that do it again. Plus, TA cloning is very easy and fast. It may take you less than a 3 days to find the right clone. Later, you can subclone into the pET all you want.