Vector keeps on self ligating - help (Feb/22/2007 )

i constructed a new vector, by deleting a 5kb fragment (lac ZYA) from the original 13kb vector, which gave me an 8kb new vector . I did it by cutting with two different RE (BamHI, StuI) then filling in with Klenow then self ligation. The result is ok. Now i used that vector to insert a DNA with two different RE (sticky ends), and im obtaining lots of colonies with no insert, and worst my self ligation plate is overwhelming with colonies.

I tried increasing digestion time, did a sequential and at one time a double digestion to no avail. I tried dephosphorylating the vector, and still it keeps on self ligating..

What could be the reason? What is the best way to solve it???

Help pls..any comments. Thanks

-arvinsign-

Are you certain the digestion is working? On both the insert and vector

Do you gel purify your vector to remove denatured plasmid (DNA that will never cut but transforms well), any uncut DNA and the bit of DNA from the vector that the digest cut out?

How old is your CIP? Perhaps it is time to buy a new vial. CIP does get old.

How much vector is being added into the ligation mix? My suggestion would be to reduce the amount of vector and increase the amount insert.

-perneseblue-

QUOTE (perneseblue @ Feb 23 2007, 05:19 PM)

whew, one of my trusted experts/bioforum hero (perneseblue) is here to save me again

thanks.

Anyway, the CIP i guess is old if not expired

. Ill take ur suggestion.ill change it. 1ul of vector (50ng) was added to the mixture. My insert is 3 ul (100ng/ul)

Thanks..ill update u as soon as i have another result. Ill try to play with the ration of the insert and vector too

-arvinsign-

the concentration of insert and vector should also be increased. Keep the

If ligation volume is 10ul, I would suggest using something like
3.5ul vector and 5ul insert, 1ul ligase buffer and 0.5ul T4 ligase

When the vector is on the large side, high concentrations help.

-perneseblue-