ligase temperature - (Oct/03/2008 )
Good morning,
what is the best temperature to use in a ligation reaction (sticky ends) with T4 ligase (Promega) using a vector of about 6 kb (pCW) and an insert of 1,5 kb?
and for how many time?
I've seen that people do from 4° to RT but which is the secret to have a good result?
Thank u.
Fralab
I have try in tempretrue room. It was successuful using T4 ligase and pGEMT easy vector.
and for how many time?
I've seen that people do from 4° to RT but which is the secret to have a good result?
Good quality DNA and don't use too much T4 ligase enzyme.
For a simple two way ligation you can use room temperature or 16 Celsius. Ligation at 4 C is generally reserved for the more difficult ligation reaction. For multiway ligation (>3 fragments), I feel 16 C is better.
I hope this info helps. If you are worried about the ligation reaction, run a small sample of the ligation mix onto a gel and check too see that the fragments have ligated. If ligation has occurred you should see high molecular weight bands.
Hi
I always go for a range of temp - I tend to set a big vat (eg plastic picnic basket) of water up at around 20C then leave ligation mix in it in the cold room overnight so that the temp gradually decreases over that time to approx 4C- it seems to work well for me and the rest of the people I work with. If that isn't possible for you I would go for 16C.
hope this is useful
I normally heat the DNA to be ligated at 65 for 5 min then cool them down at 4 degree. Then add my ligase buffer then ligase. let it go in cold water ( 16 C ) O/N or 1 hour ..I however didn't maintain the temp of the water, i just let the reaction proceed from 16 to RT. seems to work all the time.
Like pernese said, quality of DNA is utmost important. The DNA should be clean / no salt contamination and contain the CORRECT overhang. sometimes ppl digest it with wrong RE and guess what ,, no ligation .. happened to me ONCE .
I used to do 4 degree but it got both good and bad. Good because , molecule moves slower can fin ends easier, bad coz ur ligase also becomes less active at this temp.
MOst of the time ligation work in many condition , what's messing around is ur DNA or perhaps ATP degradation ?! i like to aliquot the buffer + ATP in like small PCR tube to prevent repeating thaw. One tube = one reaction .
that's how i save and maintain the success of ligation!!
We always do ligation at RT.