Ligation is carried out at varied temperatures like 16, 22, 25, 37 degrees and for different time like 16 hrs, overnight, 4-6 hours, 2 hrs, 10 mins. What among all these would work best for cohesive end ligation and successful cloning? What factors decide the selection of ligation reaction conditions out of these?
Factors that can influence on success of ligation:
1. Type of DNA fragments ends. Typically cohesive ends are ligated more efficiently than blunt ones.
2. Temperature. Temperature optimum of the most commonly using T4 DNA ligase is around 37˚С. Therefore, the best choice for blunt-ended DNA fragments is 37˚С. However, sticky ends are often too short to form stable duplex at this conditions; in that case ligation at lower temperature (4˚С) is preferable.
3. Time and amount of enzyme. Higher amount of ligase entails decreasing time of reaction. Usually, 1-4 hours is enough.
4. Lenght of ligated fragments. Ligation of contigous DNA fragments (thousands bp) is challenge. If hurdles emerge, increasing of reaction time, concentration of fragments, and so forth can be helpful.
Hi,
There are many options about the perfect ligation conditions, but you never know which temperature will work (best) until you try it. In addition to your proposed temperatures, there are some protocols suggesting temperature gradients. One easy way is to place a ligation mix in a 37C water bath in the fridge. Putting warm water into a thermal jug is sufficient. The temperature is decreasing over time. This should be done over night. This worked for me several times. Unfortunately, I do not know if incubation at 4, 12 or 16 degrees would have worked as well.
The incubation time is usually limited by the time you would like to wait. Increasing the incubation time by one or two hours should destroy a ligated plasmid. Incubation over night is very popular.
Good luck!
incubating at room temperature for overnight always works in my experience.
Hi there,
I would say the longer the better. I always run ligation overnight in the fridge with periods at room temperature right before and after. And it works fine this way so I'm not ready to change anything!
The advice from Igor, James, and Dominique about the ~4 degree reaction is correct. In addition to stabilizing the short sticky ends, however, it makes the DNA relatively stiff. This has 2 effects - making it harder to circularize (intra-molecular ligation), and thereby increasing inter-molecular ligation potentially causing a high number of multiple inserts. On the advice of my graduate adviser (long ago!), I compared 20 minutes at 4 degrees followed by 2 hours at 16 degrees to continuous 4 or 16 degree ligation temperature. The 4/16 combo gave me gave me 7x more colonies. I like James' ice bucket trick, it does the same thing!
Don t forget that ligation buffer is containing ATP and it is degradable in higher temperatures. High performance of T4 ligase is depending on integrity of the ATP in buffer. to keep the balance between T4 ligase optimal activity temperature (25 c) and ATP hydrolysis issue also cross kissing of the ends, I never have preferred incubation at 37 c; at least lets not to apply when we are facing ligation problems. I myself perform ligation at 16 c for 1 H subsequently at 4 c for overnight for both blunt and sticky end ligations.
Your result of transformation will be cleared in 2 days so why dont you keep it at 4c. I do so ie keep it at 4c for a couple of days. If your ligation is successful you can trust on your first transformation. By the way 1-5 ng of DNA per 50 ul competent cell is sufficient for transformation so I would suggest not to exceed more than 5 ul of ligation reaction. putting too much of reaction may have an inhibitory effect on transformation due to reaction ingredients and salts.
Re: Tias' 2nd question. Yes, it is usually good practice to reserve a bit of a reaction in case anything goes wrong so you don't need to start completely over (not at all necessarily just ligase reactions). New England Biolabs recommends heat inactivating T4 DNA ligase before transforming (65 C/10 min) unless the buffer contains PEG. I hadn't known this until recently. As Saber said, definitely don't overdo the amount of ligation you transform - it can drastically cut down on efficiency. The rule of thumb for chemically competent cells (calcium chloride) is don't exceed 10% of the cell volume.
very interesting question; I tried 20°C few hours (with or without PEG) 4°C, 16°C ... now we stick on 16°C overnight (or longer if you can't transform next day; if it evaporate just put some ul of water and go on). If you are in a hurry you can ligate few hours transform with 1ul then let the ligation overnight (and re-transform next day if you do not have clones...) It is true that more is not better= transform the lowest quantity (1 or 2ul ) of ligation as possible . the ratio insert/vector is important (around 2 insert for one vector molecule). the final volume of ligation too; try not to exceed 20ul (10ul is better with 100ng of vector). Remember in order to reduce the background of empty vector you can 1/de phosphorylate the vector with recombinant shrimp phosphatase (can be easily inactivated before ligation) 2/ If there is a restriction site in the vector in between the cloning sites and that is not in the insert you can digest the ligation (after inactivating the ligase at 65°C) by adding .5ul of enzyme and incubate 30min 37°C.
we ligate 100ng of vector with the quantity of insert (accordingly to have an excess of 3 molecules of insert versus vector: use the following equation: (Vi=(3*100*(bpI/bpV))/cI whereVi=volume of insert needed, bpI=length in bp of the insert and bpV=length in bp of the vector cI=concentration in ng of the insert) in 10 to 20ul and use 2ul to transform 50ul of competente bacteria at 1.10e7 cfu/ug (colony forming unit by ug of plasmide) ...
Dear Tias,
In addition to the protocols already discussed here, I have found another protocol - temperature-cycle ligation - to be surprisingly useful, particularly when I have occasionally had low levels of vector DNA after several rounds of clean-ups for site directed mutagenesis. This works on the principle of alternating between 10°C to facilitate annealing and 30°C to increase the efficiency of the ligase. The link to the paper detailing the protocol is attached.
I hope this is useful. Cheers!
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC145669/
Hello,
I have set up ligation at 16oC for 10-12hrs as well, it works fine. thereafter transform the entire ligated mixture (10 micro L) .
I remember that some blunt-end cloning worked well with initially a few hours (or even a weekend) at 4°C, and then 30-60min at 37°C (with an additional adding of Ligase) worked well: after two years of every-day cloning I was 6 months ahead of my direct competitor in a questionable CD44 lab, i.e. I finished the first isogenig K.O. constructs, but the group leader and the boss allowedtolerated even their PhD student to steal the library... So, it seems the temperature should not be that critical for even time-consuming blunt-end cloning, it should not be for "normal" cloning.
As it was mentioned above, there is "no a universal condition" that works for ligation of all gene fragments. It is good to look for an optimum condition for efficient ligation of desired gene fragments.
I have used three different temperatures for ligation i.e. 4, 16, 22 and all were fine but I have used 4 degree C for ligation because low temp. will help to ligate the insert into vector successfully.
Commercial cloning kits like CloneJet PCR Cloning Kit (Thermo) and ZeroBlunt PCR Cloning Kit (Invitrogen) use standart T4 DNA ligase and suggest to ligate for 5 minutes at room temperature, even for blunted products.
No need to waste time, unless you have very low or uncertain amounts of vector and insert, or they are not pure, or your vector has not been dephosphorylated prior to ligation.
I have used 4 degree centigrade temperature for O/N ligation and there is no problem with this temperature. It will work for all type ligation. However, in case of cloning kit you will use room temperature for 10-15 minutes also for ligation of your insert.
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