I work with metagenomic DNA isolated from soil. I isolated cells from soil, purified their DNA on PFGE, electroelution, dialysis, restriction fragmented it with BamHI (checked with EtBr staining), purified it further and ligated it into vector (the vector had been restriction fragmented with BamHI and treated with phosphatase, fragmentation was checked with EtBr staining). After plating and cultivation on LB kanamycin agar, I did restriction fragmentation of vector DNA isolated from individual clones with BamHI. I found out that every clone has only three forms of vector vector (linearized, circular, coiled) but not insert. How is this possible?
Lots of good suggestions above.
Here's one more. If you have problems cloning, make sure you include all those controls that you're supposed to do.
If you do the following controls in parallel to your experiment, you should spot where the problem is:
No insert control: full ligation reaction but no insert. Unless you get significantly fewer colonies on this plate than your full ligation, then don't even bother picking colonies.
No ligase control: ligation reaction lacking insert and ligase. If you get colonies on this plate, then you haven't digested your vector properly. This may well be the problem. Uncut vector will transform very efficiently. So if even just a tiny percentage of you vector is not cut, it will swamp any successfully ligated clones.
Colony PCR is certainly effective, but I generally aim to get my ligations working efficiently rather than having to screen large numbers of clones.
Contrary to what some people say above, I've found the ratio of vector to insert relatively unimportant during ligations. Unless I've had massively different ratios, ligations seem to be quite tolerant of variable ratios. Perhaps I've just been lucky, but I would concentrate on other factors first.
Hi,
Did you check EtBr after ligation if your inserts are actually in the vector ?
What did you use for ligation ? Maybe take another enzyme ?
Did you check your insertfragments for length, they might be too long for ligation ?
Hope you get this running
good luck
cheers
Treating the digested vector with phosphatase is never 100% effective. It will only reduce the amount of self-ligation.
Try increasing the amount of your insert (or decreasing vector amount) in your next ligation, or trying a range of vector:insert ratios.
In the future its easier to design your cloning strategy such that the vector and the insert are digested by different enzymes. This helps by preventing vector-religation (overhangs will not be complimentary) and getting the insert in the correct orientation. Just make sure the 5' overhang of the insert matches the 3' overhang of the vector and vis-a-versa. There are loads of other cloning tricks that I'm sure RG community are willing to provide. Good luck!
Hi,
I agree with Tobias. With which kit did you purified the insert? For big Fragments and vector we had Problems with some Purification Kits. Further the Ratio of our insert to your vector is important (this helps maybe: http://www.promega.com/a/apps/biomath/index.html?calc=ratio). Maybe you can also restrict a small sample of your vector in parallel with BamHI and another Enzym to be absolut sure that BamHI has work. Could be also possible that our phosphatse is not working.
Cheers
Yes, it is highly possible. Phosphatase treatment prevents re-ligation of vector but is not 100% effective.
Have you purified your vector after digestion and dephosphorylation?
Have you done a control ligation where you had no insert to your dephosphorylated vectors?
Can you use 2 different restriction enzymes that do not have compatible ends instead?
If not, the best way to avoid doing a lot of plasmid isolation in the case of using a unique enzyme to insert a fragment is to screen colonies by colony PCR.
You pick colonies and allow them to grow in liquid lb/antibiotic 20-50 ul for about 2-3 hours and then take about 2 ul and do a PCR reaction. For the PCR reaction you can use primers that anneal one in your vector and the other in your fragment , in that way only the colonies that have the insert in the right orientation will give you a PCR product. Like that you can easily screen lots of clones.
Good luck
Lots of good suggestions above.
Here's one more. If you have problems cloning, make sure you include all those controls that you're supposed to do.
If you do the following controls in parallel to your experiment, you should spot where the problem is:
No insert control: full ligation reaction but no insert. Unless you get significantly fewer colonies on this plate than your full ligation, then don't even bother picking colonies.
No ligase control: ligation reaction lacking insert and ligase. If you get colonies on this plate, then you haven't digested your vector properly. This may well be the problem. Uncut vector will transform very efficiently. So if even just a tiny percentage of you vector is not cut, it will swamp any successfully ligated clones.
Colony PCR is certainly effective, but I generally aim to get my ligations working efficiently rather than having to screen large numbers of clones.
Contrary to what some people say above, I've found the ratio of vector to insert relatively unimportant during ligations. Unless I've had massively different ratios, ligations seem to be quite tolerant of variable ratios. Perhaps I've just been lucky, but I would concentrate on other factors first.
I agree checking your controls will give you more insight as to where the problem lies. Also, which bacteria did you use? Depending on the DNA you are working with, you may need a bacterial strain that for example can successfully clone the less stable inserts. Example: Inverted or direct repeats, or GC-rich tracts are not successfully cloned in many standard E. coli strains. Just trying to think of things...
Hello everybody, I would try to answer your questions:
- yes Tobias, I checked the ligation with EtBr and there was much bigger amount of ligated than unligated vector, but as I write lower it is better get rid of these selfligated vectors. I should concentrate on the absolute separation of not selfligated vector.
- Christopher I also think that phosphatase is one of the most probable reasons for the problem of self-religation of vector, I have to find out whether it works correctly, or whether vector ends degrade after some time...
- I have to agree with Timothy our experience is that increasing the amount of insert or changing their ratios doesn´t have important impact on ligation. Ligation reaction is tolerant to different ratios of insert and vector. Thanks for the link Urda.
- I should have mentioned that I digested my DNA and vector by one different (Sau3AI) and one equal enzyme (BamHI). Booth make the same sticky ends but have different sensitivity to metylation and other conditions. I have to use two different enzymes to digest isolated soil metagenomic DNA as far as there is higher change of restriction digestion with more enzymes to 100kb na bigger fragments.
- I forgot to post here one information. I isolate high molecular metagenomic DNA from many microorganisms at once from sample of soil and try to restrict it with restriction enzymes to 100-200 kb fragments. As long as I don´t know its sequences I fragment it in this way so there are equal ends on vector and insert.
- Urda, I didn´t use a purification kit for isolation of DNA, I use separation by centrifugation, further purification of DNA (that is sealed in agarose plugs) by rinsing in many purification solutions that include detergents, enzymes, chemicals. DNA in plugs is purified by pulse field gel electrophoresis, electroeluted, dialysed and fragmented by restriction enzymes.
- I think that BamHI works properly as long as I control restriction fragmentation on electrophoresis gel with EtBr. It works properly. However, I do agree with you that phosphatase doesn´t work properly or ends of vector degrade in time. I would concentrate on this event.
- Sonia, vector has been repeatedly purified (with precipitation, centrifugation, washing, electrophoresis). It should be clean enough for ligation. This types of ligation experiments worked before, therefore it should be pure enough. Selfligation of dephosphorylated vector works with insert why shall I try selfligation without it?
- I isolate DNA with unknowen sequences and I my aim is to use it for preparation of bacterial libraries that would be used further. Therefore I need to concentrate on this problem rather neccesary for creating libraries rather than thinking about colony PCR.
- Timothy you are right uncut vector did transform very effectively. It is possible that there were traces of selfligated vector and I still have to purify vector from these selfligated traces. I agree with your opinion about importance on concentrating at ligation effectivenes and small influence of varying the ration of vector/ insert on ligation efficiency.
- Cori, I use DNA from sample that can contain DNA with high G + C content or direct repeats. I use E. coli DH5a for creating metagenomic libraries. I will find out whether they are stable enough for such cloning technique, maybe I ma wrong. Good point!
You all helped me with your advices and questions, thank you very much! I do hope that I answered your questions and would be happy to answer or consult any other idea or question.
Sonia, vector has been repeatedly purified (with precipitation, centrifugation, washing, electrophoresis). It should be clean enough for ligation. This types of ligation experiments worked before, therefore it should be pure enough. Selfligation of dephosphorylated vector works with insert why shall I try selfligation without it?
Hi Tomas,
You should always use that as a control for your ligation as it will tell you how well digested or dephosphorylated is your vector. If you don't get any colonies you should expect that all the clones that you get in your vector+insert ligation are positive. If you get more colonies in the control than in your ligation I will not even bother to analyse clones but re-digest plasmid and dephosphorylate.
best
Sonia
Hello, yes it detects this sequence G/GATC/C and restricts it where the slash symbols are.
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