There are many possibilities. Some of them are:

1. Your gene of interest didn't clone and vector has been self ligated, that's why growing on antibiotic but didn't give PCR.

2. Your primers are not working.

3. Your colony PCR protocol is not efficient.

4. If doing with colonies, may be your are taking too much or too little colony for PCR.

My recommendations:

1. Isolate plasmid from colonies growing in antibiotic medium and use this plasmid for PCR (of course after dilluting the plasmid).

2. You can use restriction confirmation of isolated plasmid instead for PCR. It will be faster way to confirm your cloning.

3. Recheck your primers and PCR mixture components.

Good luck!

If you were PCRing the entire 5 kb region, it might be too much (e.i. too long) for colony PCR. Try a shorter region (e.g. 200 bp) with one primer in the plasmid backbone and the other in the insert. Also, use a small aliquot of the ligation mixture as a positive control - you should be able to detect the product there.

Colony PCR is tricky to get right. You can get false positives from un-ligated insert unless you streak colonies of interest onto a new plate of selective media.

You can also get false negatives due to using too many cells, not disrupting the cells, etc.

My advice? Don't do colony PCR. Set up an overnight liquid culture & do a proper plasmid extraction. Use 1 primer that is in the backbone of the vector and 1 primer that in the insert. Use those to find colonies that you know have the insert. Then you can set up a long PCR & sequence to make sure the insert is exactly what you want.

As my advisor said, "You can't rush quality"

Good luck!

You should have a positive control (in this case since you're amplifying a small section of the insert your positive control should be the same PCR mix but using a small amount of the cDNA you used for ligation as template). If this control worked then your ligation may simply have failed. If it didn't then it's the PCR conditions, primers, etc... If you see a huge smear of DNA in your lanes, you may want to dilute the culture/colony sample a bit before using it as template. I generally pick a colony from the plate with a P10 pipette set to 2uL, suck up the clone, pipette it into a tube of 50uL LB medium (pipetting up and down a few times to get most of the gunk off the tip) then add 2uL of that medium (using the same tip) to a 25uL PCR reaction.

I use a similar strategy as Jean-Marc DeKeyser. Resuspend the colony in 100 ul LB (with antibiotic selection), outgrow for about 1 hour (with shaking at 37), and use 2 ul as the template in a 20 ul PCR reaction. It tends to be pretty good. If the PCR is good, then you can use some of the tiny culture to inoculate overnight cultures.

You definitely need a positive control for any PCR, especially if it isn't working well.