Ligation Help

[amcguire]

Hi Everyone!
For the past 3 months I have been trying to ligate a 1.9kb insert into a 8kb plasmid. I think I have tried just about everything and I get nothing.

I have been cutting the insert out of another plasmid by a single restriction enzyme digest. I run it on a gel and then I gel purify it. I have purified it using a kit or my homemade glasswool cloumns. I then run a bit on a gel to make sure I have a single band.

The plasmid is digested with the same restriction enzyme and then dephosphorylated with antarctic alkaline phosphatase (NEB). I also run this on a gel, purify and then check the purified product on a gel to ensure a single band. I am also comparing cut plasmid with uncut to make sure I only have cut plasmid. For a while I was only getting religated vector on my plates so I become extra vigilant about dephosporylating the vector.

For the ligation reaction I have tried various ratios of vector to insert. I have even tried maxing out the reaction with no water just all insert in a 20ul reaction. I have tried room temp 1, 2, 4 h and o/n at 4 c. I have tried buying new ligase and using different brands but it doesnt seem to matter.

For competent cells I have tried DH5a, XL-1 blues and JM109s... all that I have checked their transformation efficiency with other plasmids.
When I check my plates from my ligation I usually have NOTHING!!!! I am transforming cut vector (no ligase), cut vector with ligase and vector plus my insert with ligase

I am trying to put the insert into the pGBDU plasmid and I have it in 3 different possible reading frames so I have tried cutting out the insert from other plasmids with different restriction enzymes and cutting the vector with the same one and nothing works

I am thinking I may try blunt end ligation (I'll just fill in the ends) but in the past I find I have the more success with sticky ends.

Please please please help me!!!!
Thanks,
Ali

[Mathijs]

"I have purified it using a kit or my homemade glasswool cloumns"

Brilliant!:-D It is amazing how much money they dare to ask for some powdered glass stuffed in a column...

I don't know if it is of any help, but I once heard someone telling how he used PCR to get the plasmid after ligation. I have no experience with this myself, though maybe the linear fragment could be made by PCR first, than self-ligated. Self-ligation should give much higher efficiencies. There are some very fast proof reading Taq's (<20 sec/kb) on the market that can amplify up to 30 kb. I haven't given it any thought myself yet how this would exactly work though, maybe it was only half the story...

Is it a new vector btw, or a commonly used one in the lab, that has been used to clone fragments more often?

[pigfarmer]

Sounds like you shouldn't have any problem! I usually do a 10uL reaction without any water, that is the only difference I can see! As to the PCR option, I have never had a problem cutting something out of a plasmid and putting it into another, so that probably isn't the problem...though if you try the PCR way it shouldn't be a problem as I have inserted many PCR products.


I have a quick question about the phosphatase: I usually only do ligations with two different enzymes so I have never worried about using it, but I am wondering what are the conditions/temperature you use them at? We actually have the antarctic and I am going to need to use it shortly, and I find the NEB book a tad sketchy on the details!

[amcguire]

Hey Pigfarmer!
Here is my protocol for the antarctic phosphatase:
After the restriction digest I heat inactivate the restriction enzyme. The after it has cooled I add 1ul of phosphatase and the correct volume of water and 10x phosphatase buffer. For ex: if I have done the digest in 50ul volume using NEB buffers (if not I have in the past EtOH precipitated the digest product to resuspended in it the 10x AAP buffer and water... I am not sure if the phosphatase is compatible in all buffers and sometimes I use the feremtas fast digest buffers composition unknown) I add 1ul AA phosphatase (I do this 2x), 6ul 10x buffer and 3ul water (total vol 60 ul). I then heat it for 15 min at 37c. Then I add one more ul of phosphatase and return the sample to 37 for another 15 min. Then heat inactivate the phosphatase at 65 for 5 min. Ususally I then run it on a gel to ensure my restriction digest is complete and then I purify and use for ligation.

Hope this helps