How fresh is your ligation buffer? Is the ATP in there still ok? Which amounts of backbone/insert per volume do you use?

Hello Olivia,

I usually perform ligations at 22 °C for 2-3 h with 1 µl (5 u) T4 ligase, as recommended by the supplier. Problems could arise because :

a) the ligase has lost its activity: it should always be kept on ice or at -20 °C

b) the buffer has gone bad : it contains ATP and should likewise be kept at -20 °C. However, when you thaw the 10 x buffer, bring it to room temperature and make sure that the dithiothreitol has dissolved completely

c) the DNA you are trying to ligate has been damaged by exonuclease contamination, which has  degraded the sticky ends. Avoid gross overdigestion of your fragments; if you have to use large amounts of enzyme or very long digestion times, it could be that  your DNA is contaminated with inhibitors

Good luck,

Pierre

ligases from T4 phage work efficient at 16-24 degree Celsius. However cloning involving blunt end or small overhangs like -AT in the NdeI cut site is carried out at a lower temperature like 4-degree Celsius to increase the stability of hybridizations at the ends of vector and insert. 

Please include the two control reactions (i)  single digested vector religated to assure that all the components in the reaction are working and colonies are obtained after ligation and (ii) double digested vector religated to ensure that vectors are completely cut by both the enzymes and should not give any colony after ligation and transformation. (single cut vectors will not give any colonies when ligated with insert in some cases as the inserts are generally used in 3-6 fold higher molar concentration.

If the controls are perfect, then the ligation efficiency can be improved by the following steps which work even if it is 4 degree Celsius or 16 degree Celsius reaction.'

1. including PEG-4000 at 5% w/v final concentration to help crowding effect. crowding effect improves the chances of vector and inserts fragments hitting each other in their Brownianan motion. Setting small sized reactions like 10 microlitre also helps which contain 50 ng of vector and 3 times molar ratio of inserts.

2. Using a moderately lower concentration of ligase ( minimum 1-2 Weiss unit) and avoid heat killing of ligase after the reaction is complete.

3. Using a different transformation protocol. the protocol mentioned in the paper below improves transformation efficiency to 5 % if the protocol is accurately followed as per the article instructions. only care to be taken is the time in ethanol and that transformation should immediately follow competent cell preparation. (ethnaol-100mMCaCl2 suspension could be used at 5%v/v)

http://tejas.serc.iisc.ernet.in/currsci/dec102002/1376.pdf

4. Considering that the vector is a general purpose cloning vector of size around 3Kb and insert of size >100bp but