PCR not working

Question

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.

Those bands are 120 bp amplicons - Q5. I wouldn't trust those fainted bands - Taq.

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?

Answer 1

First you should check that your annealing temperature is appropriate for the melting temperature of your primers. (and that the other PCR parameters such as extension temperature are appropriate for your polymerase)

Secondly, although most people will tell you it's not necessary, I've found that it can helpful to clean up the template DNA just a little. For bacteria, I would boil in water (put a colony into 20-30 uL) for 5-10 minutes, spin down the cell debris, and take 0.5uL of the supernatant.

Do you have a positive control? (try to amplify some other region of the genome that you haven't modified)

Of your other suggestions, I would say that primer degradation is incredibly unlikely.

Answer 2

No one can tell you exactly what went wrong, I'd check to see that your primers are the right sequence. You can reorder them again, or reorder slightly different ones; primers are cheap, and come fast.

Answer 3

MgCl2 can also be the reason.i have tried titers of MgCl2 1.5 ,2,2.5,3 with all other conditions same and it worked