I have used this system a lot before. I think you do not have problems with the cloning but you may have problems with the PCR Reaction conditions.  Take a look at the attached paper and try to see what may missed during your reaction.  I hope this answer may help you

Article The pJan25 vector series: An enhancement of the Gateway-comp...

hey thanks ahmed :) when i do colony PCR i grind the colony in water and use it as a template and it used to work before . The funny thing is it does not seem to give even empty pENTR vector size bands .

I agree with Ahmed that most likely the PCR reaction is giving problems. Do you also use Q5 for the colony PCR? If yes better change with a normal Taq polymerase (Phusion and Q5 are not the best option for colony PCR). Moreover, a colony PCR with more than 1 kb insert is not always working well. If you have an internal primer for your insert, use it with one of the vector primers (in general 1 internal primer + 1 vector primer is  the best combination for colony PCR according to my experience). Finally, check for a suitable restriction enzyme site and check some clones with restriction digestion or isolate pDNA from several colonies and use it for PCR.

P.S. For the PCR use Tm calculated for the specific polymerase, primers and conditions. 50oC sounds like a bit low. This calculator usually is fine: http://tmcalculator.neb.com/#!/

thanks for your reply. When I do the colony pcr. I have dissolve the colony in 10ul of sterile water following which i take 2.5 ul as template. I have a feeling I am using too much template considering its a pUC plasmid and has high copy number. 

I did check the annealing temperature using the neb calculator for the M13 primers given in the kit. The annealing temperature using for Taq polymerase turns out to be 40 degrees  which is kind of ridiculous . 

If I use an insert specific primer in combination with one of the M13 primers my annealing temperature still turns out to be less then 50C . 

the M13 primers according to the kit are : 

m13 forward : 5'gtaaaacgacggccag3'

m13 reverse : 5'caggaaacagctatgac3'

I use a PCR 2X master mix from thermo . 

Could you please advice me further ? 

Reduce the template, I use 1 to 1.5 ul (similar dilution to yours but in SOC media) in 25 ul PCR reaction. Design new primers with normal Tm (around 60oC) - 19-21 bp with 3' end of 2 G or C.  One on the vector and one on the insert.

Until you do that - isolate plasmid DNA and run it on agarose gel. Your vector is 2580 bp and insert is 1500, so you will see a difference. To do that, digest the plasmids with an enzyme (from what I saw as vector map, EcoRV will be the best if it is not found in your insert).

thanks for this advice. I happened to decrease the template concentration and the M13 primers given in the kit seem to give a band of 100-150 bp but not the correct sae of the empty vector ~300 bp . I do not know why they sell them in that invitrogen kit . 

I used them for a gradient pcr. 12 tubes from 40 to 55 degrees and all of them show the same 100-150bp band . 

Maybe the primers are giving some unspecific product on the E. coli gDNA in the colony PCR mixture at these relatively low T (especially the M13F which 3' end looks quite stable).  I also often obtain dissapointing results with primers from different kits. I have no idea how or even if they test these things in real clonings. If you plan to use the system intensively, make new primers with normal characteristics. If this is a single experiment - digest with restriction endonuclease to check your colonies.

Hi, Have you ever tried growing it up, mini-preped, and digest the plasmid with NotI/AscI to see whether your insert is cloned into the TOPO plasmid? It seems to me you only tried colony PCR to check the insert. If your insert is not in the vector, results from your colony PCR will be always negative.