Top 5 reasons why molecular cloning can fail

5.Trouble with your Template
The purity and concentration of your starting materials are essential for successful cloning. When you start with plasmid DNA shared by another researcher, or perhaps stored in your lab's freezer for months or years…

4.Repeated Sequences
If your gene insert contains repeated sequences, you may end up with a mixture of many different amplicons resulting from improper annealing during successive rounds of PCR. Even close similarity (without exact matching) can cause improper annealing or ligation.

3.GC-rich sequences
GC content is critical to selecting primers and determining PCR conditions because it affects DNA stability and secondary structure. Guanine and cytosine form 3 hydrogen bonds, compared to only 2 bonds between complementary As and Ts.


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