I am trying to check the integration of DNA into a very specific region of the E. coli genome, and for that I'm comparing via 2.5% agarose gel electrophoresis the length of whole cell PCR products (I'm expecting 120 and 242 bp bands).
Unfortunately, the last 3 PCRs were very unsuccessful, or should I say not interpretable, as only the non-consumed primers were visible (+ gDNA) and no 120 bp amplicons.
The ladder I used is 2-Log 0.1-10 kb.
Here's also the recipe for one 25 ul reaction:
- 5 ul polymerase buffer 5X
- 0.5 dNTPs 10 mM
- 0.25 ul of each primer 50 uM
- 0.25 ul polymerase (I tried Q5, Taq and Phusion)
- 1 ul template (bacteria)
- 17.75 ul H20
What could have possibly gone wrong? Am I misinterpreting the results? Am I adding too much template DNA? Is it possible for the primers to degrade after several freezings? Are there too many inhibitors inside bacteria?