DNA ligations can be frustrating. Sometimes they just don’t work, for no obvious reason. My top 5 ligation tips will help to improve the efficiency of your ligation reactions and hopefully give a better cloning success rate.
1. Aliquot the ligase buffer
The ATP in the ligase buffer is essential for the ligation reaction, but is broken down by repeated freeze-thaw cycles. To prevent loss of this essential reaction component between ligations, make aliquots of ligase buffer from the fresh stock provided whenever you buy a new stock of DNA ligase. Make the aliquots small enough so that you can throw them away after use, and before you make the aliquots, ensure that the buffer is completely de-frosted and mixed well.
2. Heat the DNA just prior to ligation
When setting up a sticky (cohesive) end ligation, pipette the vector and insert fragments first and heat to 65°C for 5 minutes before adding the remaining reaction components. The heating step disrupts any vector/vector or insert/insert sticky end interactions that can interfere with the required vector/insert interaction and reduce the efficiency of the ligation.
3. Adjust the pH
The optimum pH range for the ligation reaction is between 7.6 and 8.0. Depending on how the DNA fragments were prepared, the ligation mixture may have a pH outwith this range. It is well worth checking the pH of the ligation mixtures by pipetting approx 0.2 micro-litres of the mix onto narrow range pH paper (e.g. pH 6-8). If required, adjust the pH using 0.2 micro-litre drops of 2M Tris base or HCl.
4. Use polyethylene glycol (PEG)
As with any chemical reaction, the speed of the ligation reaction is dependent on the concentration of the substrates. PEG is a hydrophobic molecule that takes up space in the reaction, effectively increasing the concentration of the aqueous reaction components e.g. DNA, ATP and ligase. PEG (e.g. PEG 8000) should be added to a final concentration of around 5-15% .
5. Add a restriction enzyme just before transformation
This neat trick can be used to combat high background resulting from uncut vector. If the vector fragment removed during the preparative digest contains a unique restriction site, adding the restriction enzyme to the ligation will selectively digest any uncut vector, preventing it form being transformed. Adding 1 microlitre of the enzyme around 5-10 minutes before transforming should be sufficient.
Digg
Stumble
Reddit
DeliciousAbout the Author
Nick Oswald
Nick is a molecular biologist who grazes in the field of biocatalysis by day and cooks up Bitesize Bio by night.
September 17th, 2007 at 10:57 am
Very usefull, tahnk you! Could you tell me where I can find more information (papers) on heating DNA before ligation? Thanks
September 18th, 2007 at 8:04 am
Hi Aitor
As far as I am aware, there is no literature on this - I believe it has been empirically observed that the heating step improves ligation, and that the most likely explanation is that the heating disrupts vector/vector or insert/insert interactions.
I certainly see an improvement when I use this step and the proposed mechanism makes sense to me!
October 31st, 2007 at 3:02 pm
[...] DNA ligase (EC 6.5.1.1) covalently joins the phosphate backbone of DNA with blunt or compatible cohesive ends (see figure on top right) and it’s natural role is in repairing double strand breaks in DNA molecules. In molecular biology it is commonly used for the insertion of restriction enzyme-generated DNA fragments into vector backbones. Commercial ligases are supplied with a reaction buffer containing ATP and Mg2+, which are both essential for ligase activity. Since ATP can be damaged by repeated freeze-thaw cycles, it is advisable to make aliquots of the buffer (see my article “5 DNA ligation tips“). [...]
December 14th, 2007 at 6:26 pm
Hi Nick,
I’m curious… how long and at which temperature do you incubate your ligation reaction? What works best for you?
I’m right now careful and do ON @ 4 °C for sticky ends… but I still get only few clones
Great blog by the way… I just discovered it.
Cheers,
Marc
December 14th, 2007 at 9:00 pm
Hi Marc,
Right now I use a 30 min incubation at room temperature, but I use a rapid ligation kit.
The problem could be with the vector prep - are you using EtBr/UV to visualise the digested vector? This can really reduce cloning efficiency. See: http://bitesizebio.com/2007/08/22/cloning-tips-vector-prep/
Otherwise, drop me an email (using the contact button above) if you want to discuss it further.
April 7th, 2008 at 8:16 am
Dear Nick!
Thank you very much for your nice comments.
You have written good comments on vector preparation and ligation. But I could not find, which protocol you are using for vector preparation !
Actually, I have problem with my transformation after ligations. Usually I get a few ( and sometimes no) colonies. Now I am working with a pET28b vector and i just wanna subclone a gene from pUC19 into this vector. After a few months of hardworking, I got only one of my mutants but I still have problem to get other mutants in pET28b.
I need these mutants for rapid purification of them by Ni-NTA column.
It would be very appreciated if you help me to solve this problem forever!
By the way, I use SAP for vector preparation and following that i pass phenol- chloroform, Diethylether, Treatment with NaCl- PEG , Precipitation with NaAcetate plus 99% Ethanol and washing with 70% Ethanol.
For gene preparation, I digest the gene following amplification, and after that I usually use a short time UV translumination for separation of the gel containing my gene and after that I have QIAGEN kit extraction of the gene.
With my best wishes,
Dadashi,
Mohammad DADASHIPOUR,
dadashi_2000@yahoo.com
s776004@st.pu-toyama.ac.jp