Hmm, when do you add the competent cells? If you are ligating 16 overnight, I'm assuming you transforming the next day, and colonies get checked for the day after that. Something like this.

day 1, set up ligation and ligate overnight

day 2 transform cells

day 3 check for colonies

What size is your vector? It's usually 50 ng per every 3 kb of vector size (so if your vector is 6 KB then you need 100 ng).

The ratio for vector to insert is 3 moles vector for every 1 mole insert.

I'd add in a transformation control too, a completed ampR plasmid (ideally your plasmid but without your insert) to make sure the cells and plates are good

here are some ligation/digestion controls

v- control, digested purified vector that you add ligase buffer, water, but no ligase and no insert, this is a check for any uncut vector

v+ control, digested purified vector that you add ligase buffer, water, ligase and no insert, this is a check for self-ligation of your vector

then have your actual ligation

Hope this helps!

Hi Yian,

I have worked with the pJET1.2 cloning kit and the ligation time for this kit for PCR-Products is 5 min at room temperature (22°C) which can be increased to 30 min for PCR products more than 3 kb.

Quotation from the manual "for PCR product >3 kb ligation can be prolonged to 30 min. Ligation times longer that 30 min are not recommend and may decrease cloning efficiency."

So even when your ligation temperature is only 16°C I would consider a ligation over night way to long. Usually the 5 min are enough.

In addition if you don't purify your PCR product you should not use more that 1µl of the PCR product as to much unpurified PCR product might also decrease your ligation efficiency (in this case you might not have the 1:3 molar ratio but usually this also works).

I am not sure which kind of competent cells you use but with these kits I would also strongly recommend to use cells bought from the company and not to use "home made" competent cells as the cells you buy are often way more efficient.

IF you still have problems I would consider changing the cloning system - eg. try the TOPO cloning kit (if this is also fine for your experiments)

Good luck

Also, did you run out any of the insert on a gel to make sure it's just one band? Primer-dimers, additional bands, etc can make it challenging to clone just the piece you want.

Did you try spreading transformed cells on a non-selective media plate? That will tell you if the cells are still viable - if yes then you see a lawn.

Are you doing chemical or electrical transformation? If electrical, any "pop" or "spark" means your cells are dead. If chemical, the timing & temperature of the heat shock are critical for success.

See if you can rule out any issues & we can give more advice.

Good luck!