10 replies to this topic

[029310]

Hi, I'm making a ligation of a 1200 bp insert into a pECFP-N1 vector but the vector always makes probably a self ligation! My finding is the next:
I digested the vector and the fragment with two different enzimes (HindIII and BamHI), and found that double digestion works well and sticky ends are not complementary. Then the fragment is ligated into the vector according to a conventional protocol. transformation was performed wirh the ligated product and also with the double digested vector as a negative control for checking the self ligation and after plating i foun the same number of colony on both the plate of the ligation reaction and the plate of negative control. The same problem happened to one of my colleagues using the same vector but different restriction enzimes (EcoRI and BamHI).
Every suggestion will be wellcome!!!
thanks in advavance for your help.
Have a nice day and a good work!

[fred_33]

hi
you can find an helpful discussion at this topic :

http://www.protocol-...?showtopic=5292

In your case, i strongly recommand you to dephosporylte your vector and to phosphorylate your insert.
Moreover increase the relative quantity of insert than plasmid. Usually i use 5fold more insert mass than mass of plasmid. But once i was forced to use up to 10fold...
Fred

Edited by fred_33, 10 March 2005 - 12:30 AM.

[029310]

Thanks for your suggestion, but after dephosforilation no colonies growth in the control and in the sample!
But the real problem is that i had just made a ligation with this vector and with the same restriction enzymes two months ago and the control (digested vector with ligase and without insert) gave me only 5 or 6 colony!!! We can't understand why now the vector make a so high self annealing even if it hasn't compatible sticky end.
Are there other control we can perform???
Thank you for every suggestion
Have a good day and a nice work

[fred_33]

hi
did you phosphorylate your insert?
after precipitation of the ligation mix (Na ac, tRNA and etoh) i usually resuspend in 10µl and use up to 5µl for electroporation. I know that if bacterias are chemically competent the efficiency of transfection is not as good as electroporation....
Maybe can you use more of ligate in order to transfect.

after electroporating, it 's recommended to let your bacterias grow for 30' to 1hour. You may increase this time?

Hope that helps you.

Fred

[029310]

I tried a lot of different ligation test changing the ratio of insert and vector, the time and the temperature of ligation, the dephosphorilation of vector and perform not a duoble digestion but two different digestion but I always obtain a self annealing of the vector and no positine ligation product.
I don't have idea if there are some other protocoll I can perform...
should be a good idea trying a ligation whit a low melting point agarose???
I'm in a huge empass, and so every help is well accepted
Thanks to everyone
Have a nice day

[george@CASE]

it sounds to me that you're digesting your vector incompletely, where only one RE is actually cutting, which will explain why you have high number of control ligation colonies and why you don't get your insert into the vector.

make sure that you are actually creating the two restriction ends. There is no way to verify this simply by running your doubly-digested vector on a gel (I assume that the two sites are on the MCS and within close proximity).

With an EcoRI-BamHI double digest, it should be done in the EcoRI buffer according to NEB Catalog. The only way I can think of checking for RE activity is to do single digests in EcoRI buffer for both enzymes, and check for linearization, which is detectable in the gel (run an uncut vector on the lane beside it).

There should be no need to dephosphorylate your vector.

Edited by george@CASE, 24 March 2005 - 12:15 PM.

[MBB08ss]

I really need some help,,, I don't know how to work this site.

I am having serious problems with molecular cloning. I have been trying to clone a PCR insert (580bp) into the plasmid pPIC9k using EcoRI and NotI restriction enzymes. I have been trying to do this for the past three months unsuccessfully. my problem is self ligation, even after de-phosphorylation for 2 hours at 37 degrees C using 1500 U Alkaline Phosphatase. No one in the entire molecular biology department of Sheffield University knows where the problem is, even though my colleagues are getting good results using the same plasmid but a different inserts.

I have run out of options, and I have no one else to turn to, not even my supervisor. It is very frustrating to have such a problem halt my entire research, for so long.

My e-mail is mbp08ss@sheffield.ac.uk I hope some one e-mail back me and give me some suggestions. I will be happy to discuss all the trails and errors I have gone through... or at least how to write in this site, and access this page again.

Thank you for your time, and have a good day.

Sari

[Beverly]

Have you tried gel purifying your cut vector? Run your digest on a gel then cut out the band and purify it. The segment you cut out will be farther down the gel and won't interfere with your ligation.

If you are ligating a mixture of vector, insert, and cut segment, chances are you will preferentially ligate the smaller piece back in, rather than your 580bp insert.

Or you need a new vial of one of your enzymes.

If this is something you have already tried, just let us know.

[perneseblue]

Hi MBB08ss
All of us here will do our best to help get your plasmid built. There are a great many things that can go wrong, so please give us as much information as you can. Write down exactly what you do, how you did it. Write down every single detail from digestion time to formulations. The more info we have, the better the change that somebody can spot what is wrong or give improvements.

1 - the PCR insert, I am assuming you are cloning it in using NotI and EcoRI. If that is so, how many bp do you have skirting the ends of NotI restriction site? NotI requires a minimum of 8bp on both ends of the site before the NotI enzyme will cut it efficiently.

2 - Given that there are problems with self-ligation, how far apart are the NotI and EcoRI sites on the vector? Did you do a double digest (how long for and how much enzyme?) or did you do a sequential digest?

3 - Did you gel purify your vector DNA after digest? There is a thing called denatured plasmid, which is plasmid DNA that has a strange conformation (due to alkaline treatment) that is resistant to restriction digest but will transform.

4 - I noticed that you dephosphorylate your vector. How do you normally do it? What are the conditions that you used? Over dephosphorylation with CIP will damage the ends of your vector making it unligatable. Using 1500U for 2hr will damage said ends.

5 - T4 ligase and ligase buffer, both of these can and do go off rather easily. The T4 ligase enzyme in particular. Is anybody in the lab who is using this ligase suffering similar problems?

When you attempt to ligate your DNA again, I strongly suggest that you run a test. Run out some of the ligated DNA mix onto a gel (using a narrow well). If the ligation worked, you should see high molecular weight bands in addition to the bands that correspond to the vector and insert.

[Nrelo]

I really need some help,,, I don't know how to work this site.

I am having serious problems with molecular cloning. I have been trying to clone a PCR insert (580bp) into the plasmid pPIC9k using EcoRI and NotI restriction enzymes. I have been trying to do this for the past three months unsuccessfully. my problem is self ligation, even after de-phosphorylation for 2 hours at 37 degrees C using 1500 U Alkaline Phosphatase. No one in the entire molecular biology department of Sheffield University knows where the problem is, even though my colleagues are getting good results using the same plasmid but a different inserts.

I have run out of options, and I have no one else to turn to, not even my supervisor. It is very frustrating to have such a problem halt my entire research, for so long.

My e-mail is mbp08ss@sheffield.ac.uk I hope some one e-mail back me and give me some suggestions. I will be happy to discuss all the trails and errors I have gone through... or at least how to write in this site, and access this page again.

Thank you for your time, and have a good day.

Sari

I really suspect that the problem is due to over dephosphorylation, 2h reaction is too long and may create blunt ends. If this occurs, no matter your vector is double digested or not, you will still get self-ligated vector.

Try to do a TA-cloning first to ensure that there is no problem when you digest your insert (there should be enough bp for the enzymes to interact with)

[swanny]

5 - T4 ligase and ligase buffer, both of these can and do go off rather easily. The T4 ligase enzyme in particular. Is anybody in the lab who is using this ligase suffering similar problems?

When you attempt to ligate your DNA again, I strongly suggest that you run a test. Run out some of the ligated DNA mix onto a gel (using a narrow well). If the ligation worked, you should see high molecular weight bands in addition to the bands that correspond to the vector and insert.

Continuing on...

You can test the ligase and the digestions quite simply. Treat some DNA ladder with ligase for ~20 minutes at RT, then run out on a gel. In a similar way take some of the insert and treat it. If the digestion has worked well, you will have a bit of monomer, as well as dimer, trimer and possibly higher -mers of the insert. If you only get a dimer-sized band, one of the enzymes has not worked properly. Similar with the DNA ladder; if the ligase is good, the ladder will shift upwards.

Some people have tried this test with the vector, both phosphorylated and dephosphorylated. The dephosphorylated version should remain monomeric, while the untreated should increase in size, but I think it's a bit of a dodgy expt, so I don't bother doing it. As others have intimated, I think you're usingfar too much AP for too long. I would follow the protocl exactly (it really can be that fussy an enzyme). Alternately, get hold of some Antarctic phosphatase; it seems more robust.