Your question not very clear: I clarify: 

So you do PCR on plasmid (1) and you find band of right size. Then you double confirm by restriction digest (2)

Then you ran the product of (1) You had band of the right size.

Usually if single digest work but double digest dont then the 2 enzyme are incompatible. did you check buffer can work both of the enzyme?

Hi,

do you perform PCR on bacteria (LB medium with growing bacteria) or with purified plasmid DNA?

If you use bacteria, you need to use forward primer from vector (such as CMV, T7 etc sequence) and reverse invert primer from insert. If you use both primers recognising insert, you will always get PCR product amplified.

Additionally, cloning might abolish restriction site (but it does not happen very often).

Or there is something wrong with one of the enzymes.

Alternatively, buffer used for double digestion might not not favour one of the enzymes.

Have you checked the compatibility among the two restriction enzymes?

So you do PCR on plasmid (in which I assume it contained insert as confirmed by PCR)(1) and you find band of right size. Then you double confirm by restriction digest (2) I found in the PCR but not in Double digestion()

Then you ran the product of (1) You had band of the right size.

Usually if single digest work but double digest dont then the 2 enzyme are incompatible. did you check buffer can work both of the enzyme? yes I checked the  compatibility. Cut smart buffer

 we do transformation with the ligated product and the isolate plamsid and did pcr with the specific primer for my insert of interest. PCR was positive and I thought that it definitely contain insert. so I treated the plasmid for double digestion and did not find any insert. I ran a positive control even just to spot the right band in the AGE

 Koraljka Husnjak .....I used purified plasmid instead of gDNA as the plasmid contains the gene for my required protein.

Hi,

you misunderstood. I just wondered what you used for PCR in order to validate insert incorporation. For that you can either purify plasmid DNA (which you did, now I know) or perform colony PCR straight on the bacterial colony (i.e. by letting colony grow in LB medium for some time and then using a bit of bacterial culture for colony PCR). In the latter case using 1 primer from vector and 1 primer from insert is better option than using both primers from insert, for colony PCR.

Koraljka

Maybe your primers anneal wrongly and the region spanned between them is approximately the same as insert. 

Anyway if it is taking a very long time for you to troubleshoot I recommend sequencing. It will definitively tell if the insert is in. May be more expensive but its worth the time saved.