how to make a 3' and 5' overhang fragment to form blund ends by KLENOW? - (Sep/25/2006 )

Hi, everyone

I really need your suggestions about "klenow" to make my DNA fragment form blunt ends.
Now I have to cut out a fragment from a plasmid, make it form blunt ends and then make them self-ligation.

However, I cut the plasmid with ApaI and HindIII and these two enzyme will make it to form 3' overhang and 5' overhang, respectively.

I applied klenow as protocol to my DNA solution(only DNA fragment that I want), then carry out self-ligation overnight.

The result is that...this way seems not work well, beacaue I run gel to check my ligation mixture, it was still at the position as my original DNA soultion was.

So I am wondering about whether Klenow could carry out fill-in 5' overhangs and removal 3' overhangs in the same reaction at the same time??

Should I carry out klenow reation overnight?? or is there any way that could help my fragments form blunt ends??

thanks your advices and helps.

I guess it should do both things at the same time.

read NEB's site for klenow.

Hi,

I think if you transform the ligation rxn product, you'll get the colonies with self-ligated product that you needed. Sometimes, ligation less than 50% efficient will give good yield of transformants.

Unless you want to troubleshoot why ligation rxn is not efficient, or blunt end filling is not efficient etc...

Cheers

Hi I got this results from a EI/KpnI - phenolization



after klenow treatment without nucleotides for 5 min at 28 C and 30 min with dNtp's at the same temperature and fenolization y got this



It seems like DNA degradation, how can I modify the protocol for get better results

Thanks