Papaver

HI Fluffy,
by using less I meant the total amount of DNA. You always have to adjust your protocol to your DNA concentrations.
Helpful therefore is: use vector concentrations of 20-50 ng; a molar ratio of insert: vector = 4:1; final buffer concentrations has to be 1x, use 0.5 - 1 µl ligase.
The final volume of you ligation mix does not have to be 10 µl. You can vary it in a way you need it. You may also read the recommendations of the company you got the ligase from.

So this time it would be better to use less vector DNA because you will need more insert DNA.
Try this:  2 µl vector (~ 40 ng)
  11 µl insert (~ 16.5-22 ng)
  1.5 µl 10xbuffer (= 1x)
  0.5 µl ligase
If you still have only 5x ligase buffer, then: 4 µl of 5x buffer, 2.5 µl of vector, 13 µl insert and 0.5 µl ligase.

You also can vary the volume for your transformation. If you want to make sure you get some colonies, do two or three transformation at the same time using, let's say 1 µl, 2µl and 3µl of DNA. When I had difficulties with my cloning (usually when I had to use some old plasmids) I used to transform up to 10 µl (for 200 µl competent cells). When I remember right, the volume of your competent cells was 50 µl. So do not add more than 5 µl (which would be 10 % of the total volume).

The problem might be that the His tag is hidden by protein folding so no binding occurs. You can try purify your protein under denaturing conditions.

Or, try to tag it C-terminally, it might bind better.

Hi,

so it can work with such low amounts,I did it once with ~ 1 ng/µl. Usually I work with amounts of 4 - 15 ng/µl and it always works fine. Just do the same protocol as before, maybe with less vector concentration (20-30 is thoroughly enough)...and using therefore more in the transformation mix.

But in parallel I would repeat the digestion using more template and set up two or three reactions at the same time if you have enough template...otherwise prep new vector. Then you would have enough for ligation.

Hi Fluffy,

negative control means you are adding water instead of template to the reaction. If a PCR band occurs you know that there is a condamination. And, often those bands are not that bright as the real positive ones...

So, if you are doing a EcoRI digestion with some other clones, wait for the result and then chose the clone that has the right size, either one of the Eco-digestion or No. two of the picture you have posted here.

So, I would say there is no insert.

Which clone(s) have you checked by digestion? Based on your PCR-Gel, I would have chosen samples 6 and 12.
One question considering your PCR...have you run a negative control? All bands besides lane 6 and 12 seem to me like they could be false positive.

Have you tried another enzyme just for checking your constructs? Are you sure that neither NdeI or XhoI cut in your insert?