Working with TOPO II cloning vector - (Oct/30/2008 )
Hi, I have been trying to insert my PCR product into topo vector and transform it into E.coli competent cells. The E.coil cells are then place on a kanamycin agar plate for incubation afterwards. After the incubation I have got 1 colony on the plate, and its quite visable (large). I inoculate that colony into LB broth w/ kanamycin. But some how, the colony of bacteria grow on the agar plate with kanamycin, but not in the LB broth with kanamycin. Why is it so?... and is there something i have to be aware of in order to have more colonies so i can get a large sample?
-Travis-
QUOTE (Travis @ Oct 30 2008, 11:59 AM)
Hi, I have been trying to insert my PCR product into topo vector and transform it into E.coli competent cells. The E.coil cells are then place on a kanamycin agar plate for incubation afterwards. After the incubation I have got 1 colony on the plate, and its quite visable (large). I inoculate that colony into LB broth w/ kanamycin. But some how, the colony of bacteria grow on the agar plate with kanamycin, but not in the LB broth with kanamycin. Why is it so?... and is there something i have to be aware of in order to have more colonies so i can get a large sample?
For TOPO cloning, you need to perform the PCR with an enzyme that generates a T-overhang. Not all polymerases give such a T-overhang. Did you use a TOPO-compatible polymerase?
-Theo22-
QUOTE (Theo22 @ Oct 31 2008, 01:09 PM)
For TOPO cloning, you need to perform the PCR with an enzyme that generates a T-overhang. Not all polymerases give such a T-overhang. Did you use a TOPO-compatible polymerase?
Just to clarify this, there are two types of TOPO vectors, one are designated "TOPO TA" and you have to use a polymerase generating A overhangs (Taq class), the other one "TOPO Blunt" needs product without A overhang (Pfu class).
Beware that some enzymes (e.g. Roche Expand) generates a mixture of products with and without overhangs. Read the polymerase specification.
-Trof-