blunt end cloning - please help me understand this... (Sep/10/2006 )
i know that blunt end enzymes like EcoRV for example cut and generate blunt ends....so if i have EcoRV in my PCR insert and now want to insert in into the vector containing another blunt end enzyme (Eco47III) it will also stick?? 
i mean the bases are not complementary so how will it work??is it that the enzyme cuts just both strands of DNA like in a straight line, leaving square like end?? 
and also if possible please let me know if i have to do something special in treating such vector before ligation. thanx alot.
Yes.
Yes.
CIP treat the vector, gel purify everything, and use more ligase and time in ligation (blunt-ended ligation is less efficient than sticky-end ligation).
thanx a lot Homebrew, i understand now. usually i use the maximum time of the ligation (30min) as written by the ligation kit system. do you recommend even more?
hi kathy,
i generally leave my blunt end ligation overnight at 15C.
thanx a lot Homebrew, i understand now. usually i use the maximum time of the ligation (30min) as written by the ligation kit system. do you recommend even more?
hi kathy,
i generally leave my blunt end ligation overnight at 15C.
Yes, so do I, you really need to increase time with blunt end ligation.
I would leave blunt end ligations for 2 hrs RT.
Yes.
Yes.
CIP treat the vector, gel purify everything, and use more ligase and time in ligation (blunt-ended ligation is less efficient than sticky-end ligation).
If you want to increse the ligation efficency, after CIP reaction you could incubate your sample (Vector, fragment ligation buffer) for 5'min at 65°C. After that, add the enzime, ATP and proceed with the ligation reaction. This allows vector to be more relaxed in order to facilitate the fragment insertion!
i generally leave my blunt end ligation overnight at 15C.
even if im using a kit where it is recommended to leave from 5-30 minutes? please clarify this point to me.
that's right, kathy. overnight at 16C is how I always do ligations, unless they're sticky and I'm in really big hurry. even if your kit says it 'can ligate' in 5-30 min at RT, that doesn't mean you'll get the best efficiency under those circumstances...and DNA ligase is DNA ligase, no matter what kit it comes with. just out of curiosity, are there guidelines somewhere in your kit protocol for blunt-end or otherwise inefficient ligation reactions?
There is a quick ligase kit from NEB. U need to incubate for only 5 min RT, even for blunt end ligations.
It has always worked when I was having difficult cloning steps.