Generally, if you follow the protocol provided by the supplier of ligase and if the lilgase has been stored properly, the problem does not lie with the ligation protocol. Causes of failure to get tranformants may be a) inadequate preparation of the DNA: impurities, degradation, ends of the fragments not compatible, inadequate quantities of vector an insert etc. b) failure of transformation due to not sufficiently competent cells. My recipe for ligation:

- 40 ng vector DNA

- A 3-5-fold molar excess of insert

- 2 µl 10 x ligase buffer (provided by the ligase supplier; make sure to warm it (≤ 37 °C) and shake it to dissolve the dithiothreitol flakes)

- Water qsp 20 µl

- 1 µl ligase 5 u/µl

Incubate overnight at 20 °C and use 2 µl for transformation

Are you using two different enzymes to cut your vector and insert? A bit more detail would help us help you troubleshoot.

I usually set up a few controls as well

1. cut vector, no insert, no ligase (this lets you see if any uncut vector is carried over, it is less of an issue if you cut with two enzymes)

2. cut vector, no insert, with ligase (this lets you see uncut vector plus and re-ligated vector, again two enzymes will prevent re-ligation)

a third tube has cut vector plus insert plus ligase (your desired ligation reaction)

Try transforming in another vector with the same selection as a transformation control, that will help determine if you have a ligation issue or a transformation issue

competent E coli keep for about 1 year at -80 and the cells do decline in effectiveness over time

Ligase buffer can also expire, check the date and the storage temperature

I would need to see which gene you are introducing, and have a little more of detail. But look for a common restriction site. Make sure that you get clear bands before ligation and increase the insert:vector ratio if necessary. I use 3 to 5 insert respect vector. Make sure

Pierre Béguin Thank you for your nice guidance.

Andrea Guedez & Amy Klocko thank you for your nice answers.

Need more information about the restriction enzymes and buffer as some of them are fast cutters and some of them are slow.

Also need information about the inactivation of restriction enzymes after digestion.

Provide information about the duration of incubation for digestion and ligation and temperature also.

Chankit Giri thank you for your answer,

I am using BamHI and ExhoI enzymes to cut the plasmid vector, and inserts gene.

for inactivation of ligase using 65 c for 10 minutes,

for ligation at 22 c for 1 hour and overnight at 16 c.

for digestion three hours at 37 c.

still troubleshooting

Please help me anyone

Thank you

Have you checked digested vector at agarose gel??