Did you run a positive control for your PCR in parallel? Ideally you should use the same primers with a control template (another vector with your insert or simply your purified insert) to rule out a problem with your reagents or cycling conditions. Did you use the same oligos and cycling parameters for your failed PCR and the colony PCR?

I would suggest checking your plasmid by restriction digest to determine whether your insert is present or not. Perhaps you had a false positive in the colony PCR.

So now I'm running another PCR with control...For PCR colony I tested 4 clones and I amplified my target. And I'm using the same oligo for both PCRs.

By restriction digest I could not cut out my insert using my restriction enzyme introduced into my olig( witch served to amplify my insert) !!!

Is it possible that your plasmid recombine into chromosomal DNA such that you can't not isolate your plasmid DNA?

Does your insert already exist in the E. coli genome? What are you cloning?

I don't, know my insert size is 1.1 kb from Rhodococcus corresponding to luxR transcriptional regulator (based on automated annotation)

Try to BLAT or BLAST your sequence against the E. coli genome to check. There may be a closely related homolog. Was your product from the colony PCR exactly 1.1kb?

yes exactly what I targeted and my oligo flanked by restriction sites

What are the restriction sites you incorporated into your oligos?

I incorporated NsiI sites in order to clone it into my vector shuttle, so after ligation procedure and transformation of EcoliS17 competent cells, I screened for positive colonies on LB pate, with appropriate antibiotic, by PCR.... it's works but after miniprep no amplification ! and I tried to blast my insert against Ecoli genome, no corresponding sequence match....

Is the problem that you recover empty vector, or no plasmid at all? I can't tell from the posts. If you are not recovering plasmid, there could be a number of different reasons and you would need to follow a different troubleshooting path than if you are able to isolate plasmid, but it lacks the expected insert.

last PCR confirmation

Are you transformation from a ligation or are you putting in the complete plasmid from one strain into another? What primers are you using? Are you checking for your insert? Can you not use M13/T7 primers or something similar, that way you should not get a fialed PCR, but the size of the product will depend on whether or not or insert has ligated in.

Be sure to linearize the plasmid before PCR - often, supercoiled DNA will hide the target - and thus it won't be readily available to the primers until thermal breakage occurs. If no thermal breakage occurs, then no PCR product. See the paper:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0009545

It looks like you are getting a band of the correct size from the plasmid DNA, but it is very faint. Contaminants in your plasmid prep could be inhibiting the PCR reaction. How are you purifying the plasmid DNA? Try spiking your plasmid PCR reaction with a "low" amount of the positive control plasmid and see if you can amplify it. If you are using spin columns to purify the plasmid DNA make sure you are getting rid of all the isopropanol from the last wash step before eluting the DNA (1-3 uL can hang up on the membrane seal).

It also looks like you added a lot more of the primers to the colony PCR reactions than to the plasmid PCRs (see fuzzy lower band in colony PCR lanes). Maybe you need to add more primers to the plasmid PCR reactions?

Jack, that is an interesting finding on the linear target.

In the past I have used a housekeeping gene/cDNA as an internal control for RT-PCR.

Jack- I don't understand your point at all. That citation shows that circular templates gave higher amounts of amplification not lower. Plus the study focused on qPCR, and not colony/insert screening. When working properly, the latter is a binary screen (positive/negative), and only meant to detect presence of an insert, not quantify its abundance.

If plasmid linearization were required, PCR colony screening would never work. Clearly this is not the case. It is true that GC-rich regions or DNA that adopts unusual conformation may amplify better from linear rather than supercoiled DNA, but this is the exception rather than the rule.

I think what the paper showed is that supercoiled plasmid DNA when used as a control was not amplified as efficiently as linearized plasmid DNA. As a result, the qPCR results for a gene/cDNA of interest was "over" estimated based on the scDNA plasmid reaction because the cDNA was of course linearized DNA and amplified more efficiently.

So using linearized plasmid DNA in a PCR reaction would give faster amplification and so more product. I have never done it, but the fewer cycles needed the fewer potential mutations in the PCR product.

Hi Gary. Yes, you are right, and this makes more sense now. Higher abundance due to normalization to a lower value. Nonetheless while linearization can't hurt, I would be surprised if supercoiled DNA accounted for the phenomenon Slimane is seeing. Will be interested to see if linearization makes a difference in his results!

I am as well interested to see if this is the actual problem or not - given Daniel's insights. My initial concern came from an absolute quantitative angle - and how that is impacted. I see now that this was not a qPCR question. And I agree with Daniel, there should have been at least some amplification if insert was present - in most cases.

It has happened to me once before that I did not get any amplification on PCR although my clones were having the insert. I had tried changing the PCR reagents. Try using a high fidelity taq ploymerase. In my case, the dNTPs were faulty. Try using a fresh kit. Maybe you have the insert but the PCR is faulty. Also, as Gary mentioned, you MUST have an internal positive control. This will tell you whether there is a problem with the PCR or with your clones.

Try sequencing your clones for insert. Just a way to determine that there is no frame shift or incorrect orientation of the insert.

Good Luck!