Hi David,

Your protocol looks ok, but there are some details missing.

How long is the incubation time of the digest? This is probably the most critical step. You should spend some hours at this point.

How are you preparing the insert? Is it digested and treated with CIP as described for the backbone? This would prevent the ligation and could explain the observed results.

Moreover, you could try other ratios of insert to backbone e.g. 3:1 or 1:1. Additionally, it might be helpful to check the transformation efficiency by using another plasmid as control.

Good luck!

Hi David,

First of all, I strongly suggests that using different restriction enzymes to digest your DNA.

I have no experience for using CIP to treat my sample before. But in your result, it seems that the phosphate had been thoroughly cleaved by CIP in 5 min. Maybe you can shorten the treating time or treat with lower concentration of CIP. On the other hand,  could you provide more information about the property of restriction enzyme after digesting you DNA (blunt end or sticky end?)

Good luck.

Weber

The fact you had many colonies suggests your competent cells and perhaps ligation reaction are fine. There may be some contaminants from the agarose gel purification. Is your insert also purified from agarose gel? 

Is it possible that CIP activity has survived and now exists in your ligation reaction?

Just some brainstorming - too many years ago that I cloned (and even won a race of 4 people in the lab six months ahead, but was not send to an experienced lab to produce my K.O. mice...), so I may miss some points here:

1) (how) do you inactivate the alkaline phosphatase?

2) I remember some little trick (but that was probably for blunt end cloning: some filling up or adding some phosphate, not sure if it was ATP)

3) some 4 °C incubation over night, then ligation at 37 °C with adding again ligase.

The length of time for digestion is probably the most crucial issue here. How much DNA and how much time?  For cloning I always go overnight at 37C or whatever the temp of your RE digests at. While that may seem overkill an overnight digestion is quite complete and I never CIP and get very few no insert colonies.  The protocol I use is similar to your. I digest 1ug of DNA overnight using 1ul of RE for a 20ul reaction.  Next day I gel purify the digested plasmid or if I have done this particular digest before using this particular enzyme with this particular vector, then I will use the PCR purification columns.  The difference is that the gel extraction gives you a very low yield in return, the PCR purification gives a much better return, depends on how much of the vector you need.  At this point you can CIP but I never do.  Regardless, I ligate using a 1:3 and a 1:5 ratio, ligate and then transform, and also include the digested vector control. The other question is, are you using the regular T4 ligase or the quick T4?  Never did get quick T4 to work well, I generally stick to regular T4 ligase.  I also never inactivate the ligase. Generally always works.  Unless there is something trickier about your cloning that you have left out.

I did the same thing to construct the library ,and I treated with CIP under the instruction book ,then transformd with CaCl2 method ,the same thing happened  ,and I used analysis and solution  is as follows:

1) The ligation efficiency was not well ,so I  try other ratios of insert to backbone 3:1 ;

2) Change the ligation volume ,from 20ul down to 5ul (of course ,the concentration both insert and backbone had been concentrated) ,and more small the ligation volume ,more the ligation efficiency ;

3) Transform with chemical conversion (your competent cell mast be good ),

    the end ---OK , many colonies appeared  but it could not satisfied my exprenments ,because I need 100000 colonies ,so I ligated 10 tubes and then puried with alcohol ,and transformd use electricity (5 tubes)

Hope to help ! ! !

First of all, the CIP is removed when you run the gel, so that is not a problem. 

You do not mention how you prep your insert. Do you cut it out of a plasmid, or is it a fragment that is made commercially? If it is a cut out fragment use as much as you can, I try to cut 3ug of vector and at least 10ug of insert in 20ul to put in one well of the agarose gel. I purify them over a column bringing the vector up in 50ul and the insert in 30ul . In the ligation reaction I use 2ul of vector and 10ul of insert, the reaction volume is 30ul and I use 1ul of T4 ligase. For control I use the same amount of vector with no insert +/- ligase to determine if the CIP and digest had worked.  I incubate at 16C overnight and transform 15ul of mixture and plate 200ul. I get clones just about every time.

If your insert is commercially made make sure the ends are phosphorylated. If not you will never get your clone.

One more thing I have had problems with in the past, make sure your T4 ligase buffer is not too old, the ATP goes off and the ligation won't work.

Good Luck!

Thank you very much for all your replies. It seems that adjusting the ratio of backbone to insert did the trick. Glad to have lots of other great advice to refer back to in the future!!!

David,

These experts have given "art" advice that does not often appear in protocols. It is important to understand the specific activities/kinetics of enzymes relative to molar templates, cofactors, temperature, etc.  Please kill/remove restriction activity and remove ligase  inhibitors.  My favorite CIP is from a cold water shrimp.  BTW,  ligase protocols are traditionally performed at "cold" temperatures to promote hydrogen bonding of short over-hangs.  Some modern ligase enzymes can help "zipper" less thermodynamic interactions.

No doubt the CIP treatment is the correct method but the 5X overage is too much. I think 2 or 3 X should do well (if at all there is a need for overage)

The origin of your insert is not mentioned. If its a PCR product that employed primers that were not 5'-phosphorylated, your insert will not ligate. You could add 5'phosphate to the insert using polynucleotide kinase, or order phosphorylated primers.

But this might not solve your problem.

As Geoff Margison mentioned above, it is important to know whether the insert is obtained by PCR or released from another vector by restriction endonuclease (RE) digestion. If the insert is just PCR amplified (and not treated with any resitrction digestion), then you have to use 5'-phosphorylated primers for the ligation to work. Blunt end ligation is as such less efficient than the stick-end ligation. So try to use the ligase that recommended for blunt end ligation.