Ligation: time and temperature - (Jun/13/2008 )
Hi everyone!
I`m just have a question:
Is it recomended to do a ligation reaction in 1-2h, 16ºC? In my lab those conditions haven`t good results...but I know it work in others labs, whit the sames enzimes and buffers. Can anybody tell me why? Thanx!
It's hard to recommend anything without knowing a lot more. Is this a blunt or overhang ligation? What is the DNA concentration? How old is your buffer? What kind of buffer are you using? What controls have you run? How do you know your results are bad? I hate trying to guess conditions and trying to answer unanswerable questions.
Hi Nori,
Phage is absolutely right. Cloning is such a tricky thing that you can't afford to take chances.
Please supply more info.
I used to keep in thermal cycler at 4c for overnight
Every man and his dog has an opinion on the best ligation time and temperature.
Here's what i do:
1. 1 hr @ room temp --> transform
2. With the same tube, O/N @ 16C --> transform
3. With the same tube, O/N @ 4C --> transform
This covers all the bases. If none of them give you positives, then you need to do a different ligation. Perform a vector only ligation as the negative control and this will give you a great indication of whether the insert is actually getting in the vector. When you have the same number of vector only colonies as insert colonies then most of your insert colonies will be empty. If you get many more insert colonies than vector only colonies then your insert is most likely in there. And don't rely on colony screens if they give you negative results because they often give false negative results, confirm the clones with a miniprep and restriction screen.
Good luck, Rob
I personally like to ligate overnight. It is about the kinetics. The slower ligation, the better it is. But I am sure it can work without overnight incubation.
Hi Again,
I usually prefer to ligate in two ratios (1:3 and 1:10). Here I am uploading an Microsoft Excel file. Just put your insert and vector size in bp. Run you insert and vector (1ul) on agarose gel for 5 min and roughly judge the quantity based on the band size.
Hope this will help. Liagation temp and time: O/N @ 16C. But sticky end ligations can be transformed after 1-2 hrs of room temp incubation.
Sorry! It says I am not permitted to upload this type of files!
Niraj
There is no definitive optimal time and temperature for the ligation of DNA fragments with T4 DNA ligase because there are too many variables involved.
The reaction rate of ligation varies with the terminal sequence of the DNA. In general, the order is as follows (fastest to slowest) :
Cohesive-end ligation :
Hind III > Pst I > EcoR I > BamH I > Sal I
A/AGCTT > CTGCA/G > G/AATTC > G/GATCC > G/TCGAC
Blunt-end ligation :
Hae III > Alu I > Hinc II > Sma I
GG/CC > AG/CT > GTY/RAC > CCC/GGG
EcoR V > Sca I > Pvu II > Nru I
GAT/ATC > AGT/ACT > CAG/CTG > TCG/CGA
The ligation rate of the restriction end of Hind III is 10-40 fold that of Sal I, and that of Hae III is 5-10 fold that of Sma I.
(http://catalog.takara-bio.co.jp/en/product/basic_info.asp?catcd=B1000529&subcatcd=B1000531&unitid=U100005325)
This Takara data hints that the optimal time and temperature for ligation will depend upon the sequences of the ends being ligated. Of course, other variables include the formula of the ligation buffer, the presence of trace levels of exonuclease contaminants in the restriction enzymes, the efficiency of the dephosphorylation and whether this process damaged the ends, the presence or absence of PEG, contaminating ethanol or dNTPs, the concentration of T4 DNA ligase, the size of the DNA fragments, salt concentrations, the commercial source of the ligase, etc.
"Most experiments use T4 DNA Ligase (isolated from T4 bacteriophage) which is most active at 25°C. However in order to perform successful ligations, the optimal enzyme temperature needs to be balanced with the melting temperature Tm (also the annealing temperature) of the DNA fragments being ligated. If the ambient temperature exceeds Tm, homologous pairing of the sticky ends will not occur because the high temperature disrupts hydrogen bonding. The shorter the DNA fragments, the lower the Tm. Thus for extremely short fragments on the order of tens of base pairs, ligation experiments are performed at very low temperatures (~4°C) for a long period of time (often overnight)." (http://en.wikipedia.org/wiki/DNA_ligase) This wikipedia entry ignores the ligation of 10-12 bp linkers at room temperature.
Interesting, never thought of that before. I'll have to read up on it, Sal I is notoriusly difficult to ligate into.
Interesting thought as well. Not a major consideration with standard insert ligations though.