I am about to start cloning an archaeal gene into an E. coli vector to use as a standard during qPCR. I am either going to use pGEM - T easy or TOPO A cloning kit. I have not done this before so does anyone have any tips or suggestions to make it as succesful as possible!
It´s an easy job, just follow the instruction of Promega. They sell a kit which includes competent cells and usually it is working well.You only have to look that you use for your PCR a polymerase which creates an A´overhang. Proofreading enzyme don´t to it. Good luck!
If you're putting a PCR product into a vector, then it is very important that you use TAQ and that you use fresh PCR amplicons because the A overhangs degrade and stop the reaction from working. Try to do it on the same day as the PCR if you can.
to have a succesful ligation process i recomend overnight incubation or if its possible a weekend incubation at 4°C. i have done 11 clonations and works perfectly with longer ligation times
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