900bp insertion into PIRESnEO,problem - (Jul/03/2012 )

Hi all,

I am trying to insert 900 bp fragment in PIREs Neo vector 5.3 kB.

My insert is digested from TOPO vector with EcoRI and isolated from gel with Qiagen Isolation Kit. Then I put my isolated fragment on gel and it was detected. So I've got a fragment with EcoRI sticky ends.

My vector is digested with EcoRI ( 1hour at 37 deg.)and put on gel and it was linearized and compared to undigested vector. So theoretically my vector was also digested properly with EcorI.

After digestion I added Antarctic Phosphatase + buffer for 1 hour at 37 deg., after it I have inactivated AP and EcorI 20 min at 65 degrees C.

Then I added vector:insert , molar ration 1:1. I performed ligation with T4 DNA ligase and T4 DNA buffer 16 hours at 16 degrees C. For control I used same ligation without insert.



When I transformed TOP10 competent cells ( not commercial). They grew in weekend at RT.

I've got 30 colonies with control and 120 with vector+insert. I've checked 30 colonies with colonie PCR and I did not found any insert, I did a control for PCR with 2 different sets of primers, so the problem should be not in PCR reaction.

Maybe I im loosing my sticky ends after isolation of my fragment from gel, but if it's the case, how does it work and how can i prevent that.

Maybe my T4 Ligase buffer has low ATP, but i still get a lot of colonies with my vector+insert. So it should not be the case. Or Antarctic phosphatase does not work, does anyone had that?

Maybe my colony PCR did not work, so I need to do minipreps..

It would be nice to hear some suggestions. Thank You

It would be better to ligate a 900 bp fragment with a 3:1 ratio. what are your concentration?
use this calculator:
http://www.insilico.uni-duesseldorf.de/Lig_Input.html

The only thing I can suggest is to repeat.

Just to double check: you treat only the vector with antarctic phosphatase, correct? You may be having trouble with UV exposure. Make sure you are using long wave UV and minimize exposure time. Think about strategies that would let you use two different enzymes at each end of your insertion next time, which makes things much easier. Even easier is to clone into a vector having a different antibiotic resistance. In general, I'd prefer to see results from low-overhead minipreps rather than colony PCR. Can you do boiling prep such as this one (you can probably leave out the lysozyme, and stop after fishing out the pellet, and just load the remaining liquid on a gel): http://bioteachnology.com/plasmid-dna/rapid-boiling-method-plasmid-dna-extraction