blunt-end ligation problem - (May/31/2006 )
I have problem with blunt-end ligation my vector (5900bp) and my insert around(1200bp)
i do blunt-end ligation with PEG 8000 and with out PEG 8000 i always get the vector when i run it on gel
if some one know how to do it the right way for blunt-end ligation please help me
the ratio (1:5) vector:insert
1 μl of 10X Ligase Buffer
0.3 μl of 10 mM ATP
2 μl of 40% PEG 8000 )
0.3 μl of 6.66 Weiss Units/μl T4 DNA Ligase (New England Biolabs)
5. Add the cocktail prepared in Step on ice.
6. Incubate at 16°C for 1 hour.
did u dephosphorylate the vector before ligation?
i think you need more time for do the ligation... blunt-end ligation need almost 24 hours at 16°C.
Did you make alicuots of buffer?... see the recommendations of NEB.
I agree with donisaid.
In my humble opinion,u can not complete the blunt end liagation in just 1h with such a low concentration of T4 ligase.
Dephosperazation is also needed.