Several reasons can be stated as follows:
Reason 1: Check your insert. If it is not blunt-end, it cannot be ligated with GetClone™ vector, as this may result in false positive results for majority of the colonies.
Reason 2: When DNA concentration of the insert is too low, the successful ligates are largely reduced in proportion, leading to an overwhelming vector background. The solution is to increase the concentration of the insert DNA.
Reason 3: If the target DNA fragments for cloning are large, such as 10kb or more, transformation efficiency depreciates after ligation. This results in fewer colonies having insertions. Therefore, it is suggested to increase the insert DNA concentration and reduce the concentration of vector DNA to re-execute ligation. It will also enhance chances to get the right clone by using higher efficiency competent cells.
Reason 4: Non-specific PCR products or/and primer dimers which might occurred during PCR amplification are supposed to compete or interfere the ligation of target PCR fragment with the vector, and thus reducing the number of correct clones. Before conducting ligation, it is therefore recommended to isolate the desired DNA fragment from other non-specific DNA by gel extraction.