Natural Science Forum / Biology / Biology / September 2006

This happened to me several times and with different constructs:

I insert a fragment into a vector. I conduct a PCR where a fragment is
amplified with a forward primer that starts from the insert, and a
reverse primer that comes from the vector itself. I run the gel and I
get the fragment of the expected length. My negative control is fine.

I culture the "successful" colony clean up the DNA and conduct a
digestion for additional verification of insert presence. The insert is
no there!!! I try  with different enzymes, that should give me
fragments of different length...and all the evidence points to the
fragment being absent.

Is it possible that I should get an amplification of the expected
length with primers that respectively anneal to the insert and to the
vector, when the insert is not inserted????

Desperately yours

Gwendolyn