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Small fragment (90bp) into an AAV backbone (4.7kb) ligation problem - I can't get the ligation (directional) to work (Jan/05/2010 )

Hi guys,
its my first time here so I would appreciate the feedback and help
I ordered a custom DNA piece (sequenced verified) to introduce a multiple cloning (90bp) site into an AAV backbone.
My insert and backbones were digested via sequential over night digestions SACII --> gel clean and HINDIII --->gel clean.
The resulting pieces were verified in a new gel.

I have tried multiple attempts with dephosphorilated and non dephosporilated AAV backbones in vector to insert ratios of 1:3, 1:6. 1:9,
1:20, 1:40, 1:80, 2:80 and 2:160.

Non-dephosphorilated backbones keep religating
Dephosphorilated bacbones produce few (1 or 2) colonies without the insert
I know that the water is clean as it is my negative control
I also know that the transformations work because I use the original AAV backbone as positive control

for transformations I am heat shocking the competent cells for 50s at 42C in volumes of about 200ul.

any suggestions?
should try blunt ligation, ON ligation, 15 degree ligation? ligate, transform and grow in media overnight before plating?



A few suggestions
try to digest it for shorter times like 1-2 hrs. Also heat inactivate them after digestion.
Heat shock for 45s at 42C.
Use 100-200ng of vector and use 1:10 ratio of insert.
if possible use cells from company with high competencies
I ligate at RT for 1 hr.
It took me a while before I could clone into AAV's

good luck !!!
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