First post here and in a bit of a bind. I have been trying to clone a 3kb gene into the topo vector. I did the pcr, added 0.2ul for taq to my 50ul reaction to add the adenine overhangs (i had used a high fidelity enzyme for my pcr), purified it, set up a topo cloning reaction using 4ul of pcr product and 1ul of vector. I got good number of colonies on carb plates but when I miniprepped and ran on a gel I only saw bands corresponding to the topo vector. Any idea what's going on? The concentration of my pcr product was 38.6 ng/ul.
Does the ratio of insert to vector matter a great deal? That's the only thing I can really think of. My mentor didn't specify the need to calculate, he just told me to use 4ul. Do you guys have any other ideas as to what's going on?