Ligation problems - (Nov/23/2006 )
Hello,
I have some problems with the ligation of my DNA construct in my vector.
The length of the insert is 3,7 kb and I ligate this in with on both sites KpnI ends in my vector (EGFP-N3 and RFP). My transformation protocol works good, but there are always not much colonies on my agar. The concentration of the kanomycine is also ok.
Also I ligate this construct with XhoI ends in a Ires-NLS dsRed vector, here are also almost none colonies.
If I mini-prep the colonies and check the insert and orientation, I had never a good insert...
The ligation protocol I use is as follow:
1 ul ligase T4 (1U/ul)
1 ul vector
16 ul insert
2 ul buffer 10x
I incubate 4 hrs at RT and then transform the product into XL-blue.
The ligation ratio is 1:3 vector/insert.
Does anybody know what I do wrong?
I dont use volumes more than 10 ul and not more than 100 ng of dna per reaction.. be carefuul with the calculation of ratios
the ligation mixture appears fine. I would only suggest that you conduct your ligation overnight at 16 Celcius rather then 4hrs at room temperature. Unless you are using a quick ligase buffer, ligation at room temperature by T4 ligase is not very efficient. And this in efficiency may compound the problem you are experiencing.
As for the problem itself....
you should first check if you have DNA in your ligation mx just before you conduct your transfection. Run a gel, (with thin narrow lanes) using some of your ligation mix DNA. THis will show if you have DNA in your ligation mix and if the ligation reaction was sucessful. A sucessful ligation will show extra large bands- ligated DNA, vector DNA and insert DNA. Run this gel and report back
Also note that T4 ligase, ligase buffer can go off. After awhile the ligase can go bad. Both don't tolerate freeze thaw cycles well. Alway aliquote your ligase buffer.
Do you dephosphorylate your vector?
If you do, be aware that overdephosphorylation will trash your DNA ends. Damaged ends means no ligation. In a volume of 50ul, 1Unit CIP, will dephosphorylate 1pmol DNA in 1hr at 37 Celcius. --- A rule of thumb.
Lastly do check that your plasmid phycially matches your map. It is very unlikely but your map could be wrong... or you were given the wrong plasmid.
A successful ligation should give you 100s of colonies. If you are getting only 10 or so colonies... treat them with suspicion. They are very likely to be false positives, especially if they take more then 18hrs to appear. Antibiotics degrades with temperature... allowing sensitive cells to grow
Finally, how are you clean up your DNA fragments (insert and vector?) Be aware that any phenol, ethanol, gel extraction buffer that gets into the DNA solution will inhibit the subsequent ligation reaction. Make sure none of the above is present in the vector mix, or insert mix. Always give a good 70%EtOH wash after a phenol step.
I respectfully disagree with my friend perneseblue. Our experiments show that sticky ended ligations are near complete in 10 minutes at room temperature. You don't want too warm a room, but 23-24C seems to work fine. Unless you are doing blunt end ligations (best thought of as a completely different protocol) you don't need, and should not use, quick ligation kits. The PEG is actually inhibitory to transformation, and while it does help in blunt ligations (a great deal!) it is not a good idea if you are doing sticky end ligations.
The points about T4 ligation buffer and care in dephosphorylation are spot on. I also recommend running the gel.
You didn't say what buffers your DNA was in. With 17 out of 20 ul of the reaction being your DNA, in whatever buffer that is, this is a major consideration. For example, if it is in TE, then you are changing the Mg++ concentration a good deal. If it is in a restriction buffer, then even more changes happen, though none of them are of much concern, with one exception -- buffers containing Triton X-100 or another detergent will trash your transformation efficiency, even in very small amounts.
You can assay the sticky ends of your DNA by doing a ligation containing only one component, and looking for the ladder of ligated product. You should see high molecular weight bands if your ends are good, when ligating both the insert and the vector independently. The gel is only readable if you heat kill the ligase -- otherwise the ligase binds to your DNA and makes it run anomolously slowly.
Interesting. I may well be mistaken.
Umm... where does you use T4 ligase buffer and ligase come from? (and to be sure, this is not ligase buffer + PEG) And what quantities of DNA is being ligated here? And how much t4 ligase is being used. 10min is a lot better then my current protocol.
After the EtOH percipitating the DNA, the PEG can be washed off by adding 150ul of 70%EtOH, and spinning down the DNA some more.
I have not noticed any anormalities whilest runing ligation mixture DNA on gels. (this maybe indicative of the different quantities of T4 ligase being used). The ligase is not heat killed.
Our standard ligations are run (on a robot, so the volume is high) in a 40 ul reaction. The reaction consists of 7 ul each of two double digested insert fragments in NEB buffer 2 + BSA; 13 ul of a plasmid backbone having a different antibiotic resistance; and 13 ul of a ligation buffer + ligase + ATP master mix. The master mix has a 20:1 dilution of the NEB ligase in Diluent A, Sigma ATP at 1 mM final, and NEB T4 DNA ligase buffer at 0.67x final. The intent here is to mix something very close to the NEB T4 ligation buffer out of components which have a variety of buffers -- with some extra ATP. We found using diluted ligase works well. If you look at the unit definitions, you will see why.
We incubate 10 minutes at RT, 10 minutes at 16C, and then heat kill for 20 minutes at 80C, although our debugging experiments show that RT works fine.
DNA concentrations are kept on the low to very low side, which encourages circularization rather than concatamers. We make the plasmid backbone by PCR and purify out the short ends after RE cutting. This eliminates background insert-less plasmids. The insert RE digests are not purified.
We transform 6 ul into 50 ul of competent cells, incubate on ice 30 minute, heat shock 1 minute at 42C, add 200 ul of SOC, and grow out for 2 hours. We found that 1 hour works well for amp and kan resistance plasmids, but 2 hours is required for tet and chloramphenicol resistance.
hi !
I have been cloning for alot of months without getting any results. This is really frustrating . Could anyone please help me out in this....
Well I have this vector which is 9kb and i am trying to clone a 4kb into this . I am digesting it with a single cut Bst XI restriction site .
I use 20ng vector and different ratios 1:2 ,1:5 and 1:3 molar ratios. I just get 1-2 colonies and they do not have the insert.
Could any one help me in this. !!!
Thank you so much in advance.
Meeta
I have been cloning for alot of months without getting any results. This is really frustrating . Could anyone please help me out in this....
Well I have this vector which is 9kb and i am trying to clone a 4kb into this . I am digesting it with a single cut Bst XI restriction site .
I use 20ng vector and different ratios 1:2 ,1:5 and 1:3 molar ratios. I just get 1-2 colonies and they do not have the insert.
Could any one help me in this. !!!
Thank you so much in advance.
Meeta
first port of call, have you checked that your BstXI site are compatible?
The BstXI restriction site : C C A N N N N N / N T G G
The N's means that you can have incompatible overhangs despite both site being cleaved by BstXI
Next... as phage434 has rightly pointed out, is any of your DNA suspended in TE, or EDTA containing solutions? EDTA is a strong inhibitor of ligation reactions. Also be aware the any residue EtOH and wash buffer from the gell column will inhibitory towards ligation. Make sure you spin your columns dry... no big blop of wash buffer on the side of the column. And that your DNA is dry. However do not over dry your DNA if you are using heat. Heating at 65Celcius for 2mins is good enough. Don't over do it.
Next, have you checked to make sure that there is DNA in your tube before doing the transformation? Run a gel. Also note that both T4 ligase and the ligase buffer do not like freeze thaw cycles. The ligase especially goes off very easily when subjected to that. Make sure the enzyme and buffer is still good. Do a PNK (Polynucleotide kinase) positive control... dephospohorylated vector + PNK + Ligase + liages buffer and you should have 1000s of colonies.
How do you do your transformation. Do you first EtOH percipitated your DNA, wash with 70% EtOH , spin down and then resuspend in water?
Are you electroporating or going by chemical transformation? How much DNA are you using?
Next, do you dephosphorylated you DNA? Also note that overdephosphorylation can damage your DNA ends. What are your conditions? How much DNA (mass), how many Units of CIP and how long?
Hi !
Thank you for your quick reply.
All the information which you gave was helpful.
How do you do your transformation.
- DO you mean after ligation ? ... Well after ligation I just heat inactivate the ligase at 65 deg for 10 mins and then from the 10ul reaction i use 3ul and do transformation into TOP10 .
- My ligation reaction is in water.
Are you electroporating or going by chemical transformation? How much DNA are you using?
- I am doing chemical transformation.
- I use 20ng vector and a molar ratio of Insert 1:3 Vector : Insert
Next, do you dephosphorylated you DNA?
- Yes I do dephosphorylate my DNA by using CIP .
Also note that overdephosphorylation can damage your DNA ends.
What are your conditions? How much DNA (mass), how many Units of CIP and how long?
- I Use 200ng Vector DNA .
- use 1U CIP
- for 1hr.
Earlier I had tried 1ug DNA for 1 hr at 37 deg using CIP of varying conc such as 0.5U/ug vector, 1U/ug Vector and 1.5U/ug vector.
But in all i still had alot of colonies.so I am using this now ie 1U for 200ng , I CIP at 37 degree.
Thank you hope you can help me further in this matter. I wanted to know if we can transform 13kb into TOP 10 by chemical method or do i have to think of another ways .. ie use electroporation and also change my competent cells ?
Thank you in advance and hope to hear more.
Meeta
first port of call, have you checked that your BstXI site are compatible?
The BstXI restriction site : C C A N N N N N / N T G G
The N's means that you can have incompatible overhangs despite both site being cleaved by BstXI
Next... as phage434 has rightly pointed out, is any of your DNA suspended in TE, or EDTA containing solutions? EDTA is a strong inhibitor of ligation reactions. Also be aware the any residue EtOH and wash buffer from the gell column will inhibitory towards ligation. Make sure you spin your columns dry... no big blop of wash buffer on the side of the column. And that your DNA is dry. However do not over dry your DNA if you are using heat. Heating at 65Celcius for 2mins is good enough. Don't over do it.
Next, have you checked to make sure that there is DNA in your tube before doing the transformation? Run a gel. Also note that both T4 ligase and the ligase buffer do not like freeze thaw cycles. The ligase especially goes off very easily when subjected to that. Make sure the enzyme and buffer is still good. Do a PNK (Polynucleotide kinase) positive control... dephospohorylated vector + PNK + Ligase + liages buffer and you should have 1000s of colonies.
How do you do your transformation. Do you first EtOH percipitated your DNA, wash with 70% EtOH , spin down and then resuspend in water?
Are you electroporating or going by chemical transformation? How much DNA are you using?
Next, do you dephosphorylated you DNA? Also note that overdephosphorylation can damage your DNA ends. What are your conditions? How much DNA (mass), how many Units of CIP and how long?
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My limited life in the lab has seen me cloning for 2 years and whilst I am starting to master most techniques I still find ligation probably the most challenging technique. I have learnt a few things about it though that may help you. Here are a couple of the important ones:
1. Each ligation is individual. Different conditions work for different inserts. Some ligations do not work after 1 hr @ room temp but work well O/N @ 16C. Some inserts do not clone at standard ligation ratios but only at high ratios. There is no secret formula that works for every ligation. The best strategy is to attempt as many strategies as possible and find the conditions that work best for that ligation.
2. Focus on making a good vector preparation. A good vector preparation will clone almost any insert you throw into it. Try creating a new vector prep and focus on improving its quality.