T4 DNA poymerase - T4 DNA polymerase (Jul/22/2005 )

Hi!!
I have some problems with a blunt ligation. I try to blunt 3' overhangs of a vector cutted with NsiI enzyme, using a T4 DNA polymerase. My insert is a blunt fragment.
I had few clones that are religated vector. I think that the T4 DNA polymerase is not more efficient: I used several protocols but these haven't good results.
Help me, please!
Thanks

-Risa-

Try using Klenow fragment. Many people (including myself) prefer it over T4 polymerase.

-Elias-

T4 has a very active 3'-5' exonuclease domain so it tends to leave DNA ends with ragged ends.

Do as Elias suggested and use Klenow.

-Daniel Tillett-

QUOTE (Risa @ Jul 22 2005, 06:49 AM)

hi risa,
i am also having same problem like you and was interested to know that has klenow worked instead of T4? many thanks.
Neesheel

-neesheel-

QUOTE (Elias @ Jul 22 2005, 09:30 AM)

Try using Klenow fragment. Many people (including myself) prefer it over T4 polymerase.

hi,
i am using T4 to get blunt end, but am not having any success.... do u think klenow is a good alternative to it? also, how long do u keep the reaction and at what temp.? As i tried keeping it for 5 and 30 mins. at 37C using T4 but did not get any sucess.
your help will be appriciated.
Neesheel

-neesheel-

Use Klenow. I wouldn't have suggested using Klenow unless I thought it was a good idea.

Daniel

Molecular biology troubleshooting help

-Daniel Tillett-