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ligation/transformation failed - (Jun/11/2010 )

Hello everybody!
I subscribed just today to this forum, hoping to find someone who can solve my little problem.

I'm trying to clone a gene from e. coli (2Kb) into a plasmid (8 Kb) that provides Cam resistance. The restriction enzymes i used, NotI and BamHI, have unique sites on the plasmid, separated by a stuffer fragment of about 2 Kb that allow clean separation of doubly cut vector from singly cut contaminants when using this pair of enzymes.

This is what I've done:
- PCR (worked well),
- restriction enzyme digestion. I have no control on digestion on PCR product, but I know that enzymes work properly on the vector, since the digestion produced 2 bands: 6 Kb (vector) and 2Kb (stuffer fragment).
- purification and gel extraction of digested PCR product and digested vector.
- ligation by T4 DNA ligase, either overnight at 6C and 4 hr at 18C. I used 1:3 vector:insert molar ratio, mixing 50 ng vector and 50 ng insert in a final volume of 10 l. To make sure that ligase works, I used it on a control plasmid digested with HindIII (cuts 3 times) and I loaded the product on an agarose gel: high molecular weight bands appeared.
- transformation. I tried either heat shock and electroporation.

At the end of all, I plated on Cloramfenicol, but I never managed to get colonies. Only transformation with supercoiled vector yields a lot of transformants, so cells are really competent.

But the weirdest thing is that when I cut the plasmid, purify the vector and the stuffer fragment separately and ligate them (thus regenerating the original vector), I don't get colonies as well, in contrast to what I see with the supercoiled.

I don't think it's a problem of quantities or size (8 Kb vector + insert), since I tried also elecroporation, which efficiency is independent on plasmid size.

Did anybody of you encounter such problems yet? If so, please tell me because I can't understand where I do wrong.

Thank you and sorry for the long post,

Andrea

-Andrea84-

Have you checked the quantities of your insert and vector prior to ligation by running them on an agarose gel? ...this is the first point i would like to recommend since people often overestimate their concentrations by just using an spectrophotometer.

You said you gel purified both fragments and ligate them and do not get colonies, since the ligase works this could only be due to:
1) as mentioend above, too low concentrations or lost of one of your fragments during gel purification proccedure (check them on a gel)

2) Degredation of your DNA and overhangs due to too long exposure to UV, try to minimize the exposure since it can really ruin your ligation reaction.

Just a few general points when performing ligations ...always perform control experiments:
1) Vector only control (just the volume of vector u use in the ligation reaction without adding ligase ...this tells you something about the amount of uncut vector in your preperation ...normally u get a few colonies on that plate ...ideally no colonies)

2) Vector+Ligase control (tells you something about the potential of your vector to re-ligate, normally just a few colonies with dephosphorylated vectors)

3) Insert only (like 1., tells you something about the purity of your insert preperation ...normally no colonies)

4) Additionally you can perform vector and insert only controls with addition of ligase to see if these two elemenst are capable to ligate with itselfe and therefore digested well with both enzymes and have functional overhangs ...you should see a real ladder starting from the size of your vector/insert when running on an agarose gel (i do this often with digested PCR products to check if both ends are cut properly).

You don't have to worry about cloning problems ...we all have them :)
What i have experienced is that cloning genes from E. coli in E. coli can be a real pain ...and is often more complicated than cloning eukaryotic sequences.

Hope you can overcome your problems soon!

Regards,
p

-pDNA-

Hi Andrea84 -- welcome to the BioForums!

A few questions spring to mind:

1. I'm not sure your T4 ligase activity check is adequate. I think you should take your vector, cut it with BamHI or NotI only, recover the linerarized plasmid by gel purification, and set up two reactions -- one with T4 ligase added and one without T4. Set up three transformations: linearized vector + T4, linearized vector without T4, and uncut plasmid. Compare the number of colonies recovered on each set of plates. This will do a few things -- one, it'll check your T4 ligase activity, two, it will demonstrate that each enzyme in fact only cuts your vector once (if you get only one band when your vector is singly digested), and three, it will check the competency of your cells and the adequacy of your transformation protocol.

2. I don't know what vector you're using, but a 2 kb stuffer fragment sounds suspicious to me -- why is it so large? Are you sure this fragment doesn't carry some gene essential to plasmid replication or stability? Are you certain this stuffer fragment has no BamHI or NotI sites in it?

3. How did you design your primers? If you engineered your restriction sites into your primers, did you include some irrelevant 5' bases? NotI in particular is a tough enzyme to use in a PCR primer -- it requires quite a few 5' bases to cut effectively (8-10 bases, according to NEB -- see here). When we're forced to use NotI in a PCR primer, we've found we must first capture the undigested PCR amplicon in a TA cloning vector first, then recover the insert from this plasmid after appropriate digestion before we can clone it into the ultimate vector. I think it most likely that this is your problem -- your PCR product is not being cut by the NotI because of NotI's need for a lot of additional surrounding bases to cut effectively.

-HomeBrew-

Yes, I usually check the presence/concentration of DNA by ogarose gel.

Regarding the UV exposure, I can't rule out it is the problem, even if I use 312 nm as wavelength and always minimize the time.

The vector I'm using is not commercial, nor has been created in my lab. However I have got the sequence and the map, so I'm sure the stuffer fragment is not essential for plasmid replication and Not-Bam sites are uniques.

I'm concerned about the Not site in the PCR product. If 8-10 bases at the 5' are strictly essential for Not to cut, then this could be a problem, even though it wouldn't explain why neither the re-ligation between vector and stuffer fragment works.

I'm going to try a new ligation with the controls you listed, at least I hope to get some more information about where is the problem.

Glad to know I'm not alone with cloning troubles.

Thank you very much!
A

-Andrea84-

To avoid further problems with restriction enzymes keep this web page in mind!

check both your vector and insert in a self-ligation reaction via agarose gel electrophoresis ...than you can easily judge if you have the right overhangs.

Good luck!!! ...i'm sure u will succeed!

Regards,
p

-pDNA-

Another good control is to cut your PCR product with each enzyme independently, then ligate just the cut PCR product. You should get lots of double length fragments if the cutting and ligation are working. This works best with heat killing the ligase, but do not use quick ligase buffer or other buffers with PEG.

-phage434-

You can also add 1 mM guanosine to the agarose gel (and the running buffer) from which you recover the fragments -- it acts as a "sunblock" and protects the DNA against UV damage.

-HomeBrew-

Andrea84 on Sun Jun 13 17:05:08 2010 said:


Yes, I usually check the presence/concentration of DNA by ogarose gel.

Regarding the UV exposure, I can't rule out it is the problem, even if I use 312 nm as wavelength and always minimize the time.

The vector I'm using is not commercial, nor has been created in my lab. However I have got the sequence and the map, so I'm sure the stuffer fragment is not essential for plasmid replication and Not-Bam sites are uniques.

I'm concerned about the Not site in the PCR product. If 8-10 bases at the 5' are strictly essential for Not to cut, then this could be a problem, even though it wouldn't explain why neither the re-ligation between vector and stuffer fragment works.

I'm going to try a new ligation with the controls you listed, at least I hope to get some more information about where is the problem.

Glad to know I'm not alone with cloning troubles.

Thank you very much!
A


A couple of things, although you've probably already solved this by now.

- 312nm UV wavelength may not be optimal for preventing damage - in our lab, 312nm is the "regular" wavelength used just for visualizing, and 360nm is the "long" wavelength used for isolating DNA. I'm not sure how much difference is makes. But, as a previous poster said, try adding 1mM guanosine to the gel and buffer. It makes things go much more smoothly, giving you much more room to make mistakes/allow inefficiencies to creep in elsewhere. (It's difficult to dissolve, though: I suggest making a 500X stock (500mM), titrating in 1M NaOH until it dissolves, then adjusting to around a 200X (200mM) stock. It may be possible to make it more concentrated.)

- If your NotI ends aren't cutting in your PCR product due to it being too close to the ends, it's not surprising that your stuffer-and-vector ligation works. Since the NotI site isn't near the end of a linear piece of DNA in the stuffer/vector, it should cut it efficiently. If anything, the fact that your stuffer/vector re-ligation is working well suggests that the PCR product is not being cut efficiently, with that NotI site as the prime candidate. If you don't want to order new primers, just do a long digestion with NotI (6-8h), and add in your BamHI for the last hour (to minimize star activity from the BamHI). It may be efficient enough that way.

-Andy Lane-