Ligation very large vector and small insert - It will not work... (Jan/29/2009 )

Hello all,

First of all, thank you for taking the time to read this. I have been trying to clone an artificial miRNA in a vector carrying the 35S promoter. The vector is 11kb long and my insert is 1.3 kb long. Both the vector and the insert were digested with EcoRI and HindIII. I have tried several approaches to deal with this: 1:3, 1:9 and 1:15 vector:insert ratios; room temperature and 16°C; 1h, 3h and overnight ligations; three different buffers; and different volumes. All these times I used 100 ng of my vector and the same ligase. You might think the ligase is the problem, but I really doubt it since it worked previously in another ligation that I did. As I type this, I am redoing it once again, with a variation, only 20 ng of vector this time and a 1:15 molar ratio. Hopefully it will work. Of course, I have to be prepared for the worst... Any ideas?

Thanks in advance for the feedback.

Pancho on Jan 30 2009, 10:30 AM said:

I have also suffered months with ligation problems and finally I completed all of them, maybe you have the same reasons of failure as me. With the ligase, it's suddenly inactive in our lab (if you were unlucky, you would be the first "victim" of that, even it worked well last time ). The second problem I got was the CIP (somehow it's worse) + incomplete digestion, even when you use two enzymes, CIP is still needed for incompletely digested vector (if only one site was cut), so I had to screen hundreds of colonies each time without success. With this problem (both control and you ligation plate has many colonies and most of them are self ligated), you can digest separately with each enzyme, purify by gel extraction, mix them and digest again with both enzymes, use longer incubation time. The third point is the ratio. Normally, I always succeed when ligating the small insert into the large vector by the ratio of 1:3, so I don't think it's your problem. The other reason might lie in your screening method. I guess you use colony PCR, sometime it works, sometime it not, so you should optimize the condition and should you positive and negative controls (I experienced that when the condition just allow amplification with higher amount of the cell, I passed many positive samples with this unexpected trouble). The last thing is the primer design, check it again for whether you designed the correct ends with RE sites and your sequence has any of these sites (once I designed primers with NdeI and BamHI sites but I was neglected to see other NdeI site near the end of my insert, so I had the similar size of insert but it has two NdeI ends and I failed so many ligations because of this stupid mistake T___T).
I hope this helps

Ok, first things first, when you say you do the 1:3 ratio, what do you mean? It's supposed to be insert:vector. You have this backwards and if you are trying to ligate a ratio of 1(insert):3(vector)...you'd be very lucky to get anything and your others are impossible. Next, when you say ratio, you mean molar, not concentration, right? I've met so many people who don't calculate the molar ratio correctly. For 20ng vector, in a 3:1 ratio, how many ng insert would you add? Should be about 9ng. Next, how have you quantified your DNA? You really need to run out 1,2,5 ul on a gel with a marker that quantifies. Compare this to what you get in a spectrometer OD260. I only run out the DNA when I have a problem and a ligation doesn't work. Turns out this week someone didn't clean out the quartz cuvettes and I got a way too high reading. Tryed to ligate about 1ng vector to tons of insert. Finally, do you (or members of the lab) freeze/thaw the ligation buffer multiple times? This buffer contains ATP which will degrade and is neccessary for enzymatic activity. This buffer really should be aliquoted so it doesn't freeze/thaw more than 3 or 4 (maybe 5) times.

Hello

I routinely clone large fragments in the lab and they are problematic to clone. This works for me:

For the vector (large size):
1. Ensure complete digestion, use less DNA and perform multiple digestions so that we can pool them later and get good conc.
2. CIP each vial followed by column purification (don't run on agarose), while purifying, pool the mixture.
3. Elute in water, check conc. and use for ligation at different ratios.

U willl get a number of colonies

4. Screen multile colonies (I generally streak each colony and inculate 3 colonies in 1 vial), so with one miniprep, i screen three colonies.

5. wherever i seeteh expected band by restriction digestion, i go back to teh later and inoculate those colonies independently.

This always works for large fragments.

Hope its works for you too.

Get bck to me if u have any problems.

Best
TC

If you are convinced of the digestion and the concentration of the fragments, then I suggest trying to use 200ng of vector and different ratios of insert, make sure the salt levels are low.

Well, I am happy to announce that my ligation finally worked. What did I do? Simply use less DNA (around 30 ng total) which game me more than enough transformants. Oh yeah, I also changed my ligase, most likely that was the problem.

Dear all,

my vector is pET22b and i am digesting it with Nde1 and BamH1. The size of the vector is around 5.5kb and my insert is 1.5kb. I am using 200ng vector and 162ng insert, which is a 1:3 ratio of insert to vector. Is that alright?

jiajia1987 on Feb 25 2009, 02:52 AM said:

I would start with 20ng of vector and 1:3 ratio of insert. I would use 200ng of vector for ligation as one of my last options.