Well...are you doing a miniprep or a midiprep to isolate the plasmid from bacteria? It seems like the concentration of the template plasmid you are using is too high and therefore it is showing up even after PCR...although in the PCR reaction, the concentration of the template should be pretty low...anyways, try doing a serial dilution of your template (miniprep samples preferably) and use it for PCR...as colony PCR gave you good bands of desired size, I am quite confident that template might be too concentrated when you are running a PCR after plasmid isolation...also try cutting the plasmid singly with a restriction enzyme to make it linear and then use it as template...sometimes, this works better...hope this helps...thanks.

Yes, I'm using Plasmid miniprep kit for plasmid isolation. Thanks for your reply I'll try your method. 

Do you used the right restriction enzyme, may be the enzyme not cut the plasmid at the site of your insert

Dear Nadia

You can use around 1ng even less to perform PCR on plasmids. Anyway it is strange that you say the sequencing result was not good. Normally if you have the positive colony pcr you should also see the band after plasmid extraction by PCR unless something happened during your miniprep?

Good luck

Ur welcome...gd luck...

Dear Dr. Tarek

yes, i have used the correct restriction enzymes. from 3hours i did dilution for the recombinant plasmid template as Dr. Anirban suggested and did PCR, i got  band at the place of vector site but the band of the gene appeared this time. the thing which confusing me that i did digestion also and i should get 2 bands but i got only the vector band :(

Dear Nadia,

I would suggest to check the presence of the insert in the plasmid and the presence of the right restriction sites. For this linearize your empty vector and plasmid with insert with each of the restrictases that you used in the cloning but in separate reactions (4 reactions in total). Separate products of digestion in the 1% agarose gel. 1) If your plasmid does have an insert that you see that it band goes more slowly than the band of the empty vector. 2) In case that, one of the sites is disrupted during cloning - the plasmid will be not cut and goes in the gel as the supercoiled plasmid with other minor forms of the plasmid. 3) you may get the vector-dimer, in this case the plasmid with insert should have a dobled size of the vector (it can be seen by comparing the DNA ladder).

If you get the case 2) you can further check the presence of the instert by the restriction digestion. Use restrictase that cut only once inside the insert and the other restrictase that cut once in the vector backbone. You can easily choose those enzymes and calculate of the sizes of new restriction fragmens using map of the desired plasmid built in the software such VectorNTI, or Unigene (free software). If the sites for the corresponding enzymes are intact that you will see the second band  that tells you the presence the insert.

I some times get into the 2) case when use in cloning NdeI or pair HindIII and XhoI.

Also check the compatibility of the restriction sites. For example, XhoI and SalI generates sticky ends that can be annealed but the new site cannot be cut anymore by this enzymes.

Check carefully your primers may be they have 1-3 mismateches with vector part, so they can work in the PCR conditions you use.

Good luck!

So I'm assuming you took some of the ligation reaction and digested it and ran it on a gel along with control reactions and undigested (a no DNA except primers, and a cut vector only to check self-ligation, and uncut previous vector). You should be able to tell what is what by this before double-checking for your gene by PCR. If you did not do this, you should, before checking by PCR. There will be all kinds of things in your ligation reaction still so your newly ligated vector will have to be cleaned up before you can check by PCR as well to make sure it is your gene fragment in the vector. If you don't, you may get your gene, but your mix will still have other elements in your reaction mixture such as cut vector that may or may not re-ligate. You can run your full ligation mix and gel extract correct band and then transform..etc..etc, or you can use a miniprep column, which will only grab vector DNA of a certain size and eliminate others..could use a combination of the two, or other methods. Just make sure you've isolated it before re-transforming. I hope this helps. Also, keep in mind that if you RE digested your product that the insert is much much smaller than the rest of the vector and so may not show up well on a gel unless you use a lot.  If you already did all of these things and it still did not work then sorry I didn't help much. Good luck!

Dears Dr. Arefeh, Dr. Dmitry and Dr. Cori  

Thanks a lot for your answers and your kind help, I'll try your methods and I'll let you know about the results :)