you didnt mention the amount of insert you used in the reaction. usually for ligation reactions its preferred to go for 1:3 molar ratio vector : insert. another thing is try to make your total amount of DNA in the reaction to 100 ng this will help alot. you can use this calculator to make things easier for you http://www.insilico.uni-duesseldorf.de/Lig_Input.html the link is also attached. I hope this will help you

http://www.insilico.uni-duesseldorf.de/Lig_Input.html

0 colonies means the transformation is at fault.  

If you use high efficiency competent cells and do it correctly, there will always be "background" colonies, like uncut vector, random side reactions.  Usually there should be 50 to several 100s of these background colonies.  And then, if your ligation works, there will be correspondingly more that contain your correct vector.  

There's hardly a point troubleshooting your ligation, if you can't see the background colonies.  You need to bring those up, before you can even begin. 

Hi

1) In transformation reaction always use a positive control, that will tell you either problem is in ligation or competent cells.

2) i don't see any purpose of using CIP here as you are using different restriction enzymes. 

3) i assume that u are also talking about pcdna 3.1, is it 6500 bp or approximately 5500 bp? 

When things aren't working controls are important. You should include the following to troubleshoot. 1. Uncut vector to check transformation. 2. Cut with just 1 RE and do plus and minus ligase reactions to check the ligation. Good luck!

Also, make sure the antibiotic in your LB plate is a correct one. A wrong one may kill all the cells.

A positive and a negative control helps in this case to make sure what is going wrong. You can use 1:5 (v:i) equimolar concentrations which might help.

Good luck. 

Have you tried checking the compatibility of your cells with some uncut vector of known concentration?

It also wouldn't hurt to gel purify your PCR product, to get rid of unspecific products (they are probably created, even if they can't be seen as bands). This shouldn't be the reason for an empty plate, though...

Use Positive control, 1:3 I/V molar ratio 0.5ml SOC verify antibiotic match between competent cells and vector vs. your plate 

When you do a ligation, make sure the DNA concentration is between 5 and 10ng/ul.  Higher than 10ng/ul will decrease the efficiency of the reaction.  So, you probably should quantify both vector and insert after purification. 

Also, try 1:3 or even 1:6 V/I molar ration.  You want to make sure each vector piece can find an insert.

Last, after you heat shock, return your cells to ice for 15 to 30 min, and after that add 500ul of SOC and shake for 1 hour at 37C.  It's crucial that you return the cells to ice after the heat shock.

Check out the publication in the link.

Article Plasmid DNA transformation in Escherichia Coli: effect of he...

The other thing to check is that your PCR product is being digested correctly.

An error I made once, was not ensuring I had 6bp on the 5' end of the restiction sites on my PCR primers. Consequently my restriction digests were of poor efficiency, thus affecting annealing via sticky ends (or lack thereof.....) for subsequent ligation.

I had similar problem couple of years ago. Of course including some controls would help to decide what is happening. But what solved the issue for me back then was re-precipitating the PCR product with vector together over night. You mix appropriate amount of cleaved product and vector in the ratio you like to use, add 4 ul of sodium acetate, 100 ul of Ethanol and place into -80 overnight. in the morning you just centrifuge and dissolve in amount of water you wish to have in the ligation reaction. add enzym, buffer and ligate for 16 hours. 

Also, it might be beneficial to incubate the vector with alcaline phosphatase in case you didn´t do so. It would prevent the empty vector to ligate back together.

You may use picomole end calculation for digested plasmid and insert.

Have you checked efficiency of competent cells?

You didn't mention volume of ligation reaction. 2ul ligase is much more. 

overnight incubation at 4 degree celcius can also be tried. however, I got no. of colonies after 2 hours incubation at 22 degree celcius. 

1. the concentration of cloning vector to digested DNA fragment from donor is 1:3

2. add 2ul of the enzyme and incubated at 10 C for 6hrs or at 4C for overnight 

3. transform ligation mixture into recipient cells 

Try doing overnight ligation at 5 degree C. Be gentle while mixing the content. Use pipet tip to gently swirl for mixing.

I would cut with each restriction enzyme using separate reactions, with DNA cleanup between cuts.  I would use 50ng vector, V:I, 1:3 and 1:5.  ligate reaction overnight at 16 degrees C. Also cut a portion of the vector DNA with EcoRI and a portion of the vector DNA with HindIII.  Ligate these single cuts as separate reactions to use as positive controls for ligation of each enzyme cut. Do one transformation per ligation, that is four separate transformations of four separate ligation reactions (vector/Hindiii; vector/EcoRI; V:I, 1:3; and V:I 1:5). Lastly use the uncut intact vector as a control for transformation, in summation, four separate ligations and five separate transformations.