Ligation with T4 DNA Ligase - (Jul/23/2007 )
I have a question about ligation and a problem in cloning.
I am trying to make a tandem array in pUC18. The restriction sites I use, are SalI on one side XhoI and BamHI on the other side of the insert. What I do, is basically to cut the insert from the vector with SalI /BamHI and insert to the same vector cut with XhoI/BamHI. SalI and XhoI restriction sites are complementar. So, at each cloning cycle, I double the length of the insert.
Now I am on the 5. cycle and the insert is about 1800 bp. However, I got problems at cloning on the 6. cycle. I am sure that the insert is cut out and the backbone (pUC18 with the insert=4500bp) must be cut as well (it worked the 5 times before). After DNA extraction from the agarose gel, I run 2 ul of the on a gel. The sizes and amounts look fine (insert 1800 bp and the backbone 4500bp). I ligate with 1 ul T4 DNA Ligase in 10 ul ON at 16C. I get 20-30 colonies (based on 100ng backbone). I set small ON cultures from those and do miniprep and check the presence of the insert by restriction with BamHI/SalI (in this case the insert must be 3700bp). However, all the plasmids I get, have got only a band at 2680 bp (linearised pUC18). It seems that I lose the whole insert during the ligation (even though I would have self-ligation, the backbone vector must be 4500bp).
Does anybody have any idea about that? I am open for all suggestions and would be really happy for any help.
Thanx in advance
If science was an RPG
A case of mistaken of identity..... Get thrown into Town Jail. (Prestige penalty).
The 1.8kb insert might really be 2.6kb pUC backbone. Was the correct ladder used.
Vector not ligating; Cursed by CIP! .... Lost of hair. (Charisma penalty)
The colonies you are seeing come from residual vector DNA carried over from the insert fragment. Might the vector be damage from over dephosphorylation? 20-30 colonies from 100ng DNA... isn't that much. Was there a vector + PNK ligation plate? Could you run one?
Recombination beast rears its evil head..... Go begging for new e coli strain on bended knee.
What kind of e coli strain is in use. Occasionally people make the mistake of doing their cloning work in cloning strains... which don't handle tandem repeats well. Although some cloning strains aren't too happy about tandem arrays either... but you tend to see something, truncation products... so probably not the case.
Unsolved Mysteries.... Lost of life force. Life bar shorten by 1wk
Just religate again and run a sample of the ligation product on a gel. If the ligation is working you will see the presence of high molecular weight. If gel shows a ligation failure, consider buying new ligase or using some ligase from a friendly lab next door that is known to work. If new ligase doesn't help, start from the begining and re digest your insert and vector. Sometimes starting again solves the day, especially if you are confident that the method works.
thanx a lot for the ideas!
I digested the plasmid from which I am cloning (pUC18+1800bp insert), run on a gel together with the digested "well wrong" clones. The backbone and the insert fragment are clearly separeted. I am sure about the marker (2-log DNA Ladder if you heard about it).
I do not dephosphorylate because the restriction sites (BamHI/XhoI) are not complementary.
I use regular chemical competent DH5alpha from Invitrogen (I thought it could be the bacteria). But since it worked the first 5 cloning rounds before, I do not understand what is wrong now.
I tried with new enzyme and buffer yesterday. No hope! All I have got are clones with an insert less than 1800 bp (how can the insert in the backbone become shorter?).
I'll try with religation to be sure that the enzyme is all right.
I want to keep you updated about my cloning (if somebody is still interested in that).
So, after I tried different amount of ligation mixes (10-20ul), different amount of DNA (50-150ng), different temperatures (16, RT, 37 degree) and incubation times (2h-ON) and finally three-pieces ligation, I realised that the problem is not the ligation itself but recombination.
I am trying to multiply a 33bp sequence in pUC18 vector (like a tandem array). Now I have 48 times this insert (1.8 kb). In all my minipreps I was getting a shorter insert than what I have got in the backbone. It seems that the larger the insert gets, the more unstable the plasmid. I was using DH5 alpha which is recA minus. Then I have switched to NM522. No luck. Now I got an aliquot of SURE bacteria from the next lab. I tried to make them chem. competent by Inoue method but they seem a bit sensitive (to shaking at 225rpm, for example). So, I am not quiet sure whether they become competent at all. But I'll see tomorrow.
After I have googled many protocols about cloning repetitive sequences, I know that I have to find the suitable strain on my own. Growing bacteria at low temperature might help...
I want to update my cloning experiments. So far, I have made 3 more strains (JM109, NM538 and Q358, I have got them from another lab; Inoue method does not work well for NM strain) competent and checked whether they produce my original plasmid at all.
So far only the JM109 did not show any recombination. However, the plasmid yield from miniprep was very low and I was afraid to grow them longer due to increased possibility of recombination, mutation... Although the bacteria was stacking again at the bottom of the tube (2 ml culture from a single colony in a 15 ml Falcon tube, 12 h at 37 degree, shaking at 250rpm). Now I am doing the transformation of the next cycle. In two days I will get results. I really hope that this is the right strain. Well, I still need one more cloning cycle, though
If things are giving trouble, drop the temperature down to 30 Celsius. And if you are worried grow said cell in SOC medium. The medium is much more expensive, but the plasmids you get are more intact compared to those obtained from cells grown in LB.
Oh, and grow your cells in larger volume. Try around 4ml in a 50ml tube (with a hole punched in the cap by a red hot needle. E coli need oxygen to respire.). That way you have more cells to process and avoids stressing the e coli from growing in a high density culture.
thank you for the help!
I have got now the results .
I picked 20 colonies, growed ON in 2ml LB, did miniprep, digested with BamHI/SalI to see whether the desired insert is there. So the pUC18-backbone is there. In original plasmid the insert is at 1.8kb but the prepared clones have inserts shorter than this (veri little plasmid yield).
So now I will follow your instructions .
Do you have any more ideas if these wouldn`t work (I mean as a backup plan)?
unfortunately this is the best I have for culturing conditions.
Sadly I would then suggest that it is simply not possible maintain the a 3.7kb tandem array in pUC18. Small as it is, pushing pass 1.8kb has destabalised your array in pUC18. I would suggest moving your array into a single copy BAC, like e3.6.
Being single copy and a BAC, the array should (hopefully) be stabalised.
I am just wondering about... Let`s say it works in BAC system. I wan to make a stable cell line containing this DNA. In my lab I think they use pSG5. I would need to clone my insert to that plasmid at the end. How am I going to achieve it? Do you have an idea?
If the array is stable in a BAC, I would attempt to
-find a BAC that already contains the necessary SV40 promoter, betaglobin-intron and pA for eukaryote expression work.
-failling that, I would move the eukaryotic expression segment of the pSG5 (Promoter-SV40/SV40ori-Betaglobin-intron-PT7-multiple cloning site-SV40pA) into a BAC. Then clone in the tandem repeat into said modified BAC.