Did you phosphatase treat your vector after you digested it, prior to ligation?  

Did you screen 12 colonies from both the 1:3 plate and the 1:6 plate, or did you screen 12 colonies total from both plates? 

To find your problems, you should do some steps:

- Check your medium and make sure you added right antibiotics for your vector selection

- Make sure you have a good ligation (vectors, DNA, temperature....)

- Which kinds of competent cells did you use? check the quality of competent cells and transformation conditions (temperature, time ....) by using an undigested vector like a positive control

- You should use X-gal to select ligated colonlies (blue and white selection)

Good luck!

Hi Samantha, I did not phosphatase treat my vector and used it directly for ligation after digestion. 

I picked 6 colonies from each plates (1:3 and 1:6), and today I have selected 12 more colonies from 1:3 plate (all the colonies except one) and kept it in the broth for overnight culture. 

Do you perform colony PCR and/or mini-prep purify the DNA from the colonies for RE digestion? 

Hi Phat Tien, 

I am using LB broth and the kanamycin to culture bacteria (DH5-alpha). I have used different controls: 1) Uncut plasmid + bacterial cells (positive control) to check transformation efficiency, 2) Just bacterial cells (negative control) 3) Digested Vector only (with ligase) to check self-ligation 4) No ligase control (just digested Vector) to check undigested vector. 

It seems to be working fine but I am still not getting the desired clone. To make things more complicated, my vector and insert are the same size (4.7kb each), so I have to cut it with a different enzyme for which Insert has 2 sites and Vector has 1 site in MCS.

If none of these apply, I can try to take a look at your sequences/maps and see if anything obviously wrong jumps out at me. 

Anshuli - I would suggest phosphatase treating your vector.  DNA digestion generally leaves a 5' phosphate group, which is needed for ligation. Because linearized vectors are very long and flexible they have a tendency to self-ligate. As a result you will get much higher background and colonies that don't contain your insert.  Phosphatase treating is really easy, just add a 1 ul of your enzyme and incubate for 10 mins. This will prevent religating of your vector.

It's possible you were just unlucky and picked colonies that didn't contain your insert.  Six colonies from each plate is not many.

After transforming my bacteria with my ligation product and plating overnight, I will do a colony PCR.  Depending on how packed the plate is, I will pick between 10-20 colonies to screen.  For my forward primer, I use a T7 or SP6 (whatever my vector contained), and for my reverse primer, I use the same reverse primer that I used to clone my insert.  Based on PCR product size and presence, you can determine whether the colony you picked contained your insert.  I attached a protocol from addgene on how to do colony pcr.  

I make sure I know from which colony my positive PCRs came.  Then I culture one of those positive colonies overnight. The next day, I do a mini prep, and send the DNA for sequencing to confirm the presence and orientation of my insert.  

Hope that helps.  Good luck!

https://www.neb.com/products/m0371-shrimp-alkaline-phosphatase-rsap

http://blog.addgene.org/plasmids-101-colony-pcr

http://www.csun.edu/~mls42367/Protocols/Colony%20PCR.pdf

You had only few colonies even from positive control vector. So, I still have a concern about competent cell quality and  ligation, transformation procedure. Normally, I use 16oC overnight for ligation. I use SOC medium for growth E,coli before spin down to have bacteria pellet to spread on LB medium.

plate non-transformed competent cells on kanamycin plates and check if the kanamycin is working. you should not get any colony.

I agree with Samantha! You need to dephosphorylate the 5'end immediately! you do not want vector re-ligation. Also, make sure you have digested your plasmid properly!