PCR may generate mutation i mean the DNA polymerase is known to induce or create a single base pair mutation which can lead to truncated sequence of DNA in this case try to optimize your PCR reaction for instance if you amplify the sequence that contains GC rich regions add betaine to your reaction. Or perhaps during the stage of digestion by restriction enzymes have you checked if you have internal cleavage sites ? More details are needed good luck

check your PCR product by DNA sequencing

well.. first you check  whether the digestion and ligation of your vector and insert are complete or not?? you might be getting the colonies after transformation due to incomplete digestion of your vector!! Secondly, are you using any selection marker or not? if yes, then the transformed colonies are result of incomplete digestion of your vector. Please follow the digestion and ligation protocol cautiously.

Good luck!

Some DNA fragments are simply not stable - either because they contain big repeats or because they produce lethal products. A sign of this would be unexpectedly low yields of recombinants and/or only one orientation of the insert observed and/or colony size variation when you spread recombinant colonies. 

For long fragments I advice you to use the Pfu polymerase, that introduces less mutations than Taq polymerase.

Try to do also some mini prep of your colonies and digest them using as a negative control the empty vector.  When you amplify on the colonies very often you can have different products by PCR...due to not optimal melting temperature of the primer. 

Do cracking gel and then restriction digestion to see if you can recover the insert and vector band. 

May be you have multiple bands. Try to separate well agarose gel (using long gel) or elute using 7% PAGE.  After elution, ethanol precipiation helps to increase ligation efficiency. 

Besides using a longer gel to really get the right band you can maximize the number of clones you test by searching a larger number of bacteria. To do it pick a small amount of each colony in 10 microliters of water and use 2 microliters as a template in your PCR.

Sometimes Colony PCR is not the best way to check your product. If you have a large fragment it can break into couple of pieces and you can see several bands once you run the gel. Sequence your DNA, this is the best way to verify your products.

Some time bacteria can modify your DNA sequence following transformation. You can try to use other strain as Stbl2 or you can use a "new way of cloning" called InFusion. I did personally use InFusion and it worked. Once you have your gene into the vector you can transform the strain you need.

hi!

did u confirm your clone post ligation  by restriction enzyme digestion?

you can also go for confirming your exact insert by using the same set of primers and using the complete construct (vector+insert) as template and doing a PCR. you should ideally get the specific amplification of your target gene.

then you can ,move on to next step..

Thank you very much to every one for your valuable suggestions am trying to standardize my PCR protocol once again and i will go through my protocol according to the above said measures 

When you  say "You are cloning your insert into a vector", did you mean that you are performing directional cloning? If yes, then have you checked if the RE  site that you have chosen is NOT present anywhere within your gene?

More information would definitely be helpful

Hello, from your question, I understand that, in your elution you have atleast two products. One is smaller than the actual gene of interest. In that scenario, I recommend you to elute the band of interest by using 7.5% PAGE due to its better resolution (if amplicon size is less than 500bp. If no reduce PAGE concentration).  Secondly, screen atleast 20 colonies by colony PCR.  Good luck.