ligation problem.. - (Apr/24/2005 )

i got problems here..plz help!my vector is 18kb while my insert DNA is about2.5kb.i done the ligation with PEG and it happened to fail to be transformed..whats wrong?i ,mean,is it the sizes of vector and insert gave me the ligation failure?

Hey,

The size of your vector is "uncomfortable" but anyway it is not the reason that your ligation failed.
How do you purifie your vector after restriction?
How do you purifie your insert after restriction?
The problem might be a purification with phenol-chloroform.
It may be worth trying some unusual ratios between vector and insert as well considering the size of your vector.
Do you know that your ligase is working properly, are your bacterias good?
You can test your ligase through ligating a DNA ladder and put it on a gel for example.

Hope this is some input
Cheers

hi
usually, ratio Plasmid/insert is 1:3 o 1:5. But if you're facing problems, you can try to force the balance in your advantage by increasing this ratio. Once i only obtain my clone by a 1:12 ratio...
Did you check your ligation? I mean when i do a ligation with 100ng of plasmid, after the precipitation/resuspension of DNA in 10µl i usually check 5µl on agarose gel to see what it looks like, comparing the ligation with negative control. It's not a strong way to check, but if you're able to see difference, it raise the possibility that the electroporation step may be a failure...