Cloning big plasmids / triple ligation - hellpppppppp (Aug/26/2009 )

Hi there!

Someone here with experience in cloning big plasmids?

I have a piece of 3.3kb to put into a 14kb vector. I tried to clone this piece before in a small plasmid (3kb) and only worked when I cut this piece in 2 : a 1kb fragment and a 2.3kb fragment. Initially this 2 pieces where separated by a neo casette but after removing it I was never able to work with the 3.3 kb - entire piece, I guess because I have some repetitive sequence there.

Anyway now I want to try a triple ligation :
Pac1::::::1kb piece::::::Bgl2
Bgl2:::::::2.3kb::::::::EcoR1
EcoR1:::::::vector::::::PacI (14kb)

Did anyone try something similar or has a recommandation for how to make this to work?

Thanks a lot!

If you have good DNA fragments with intact ends, this should just work. Use equimolar amounts of each DNA fragment at relatively low concentrations (10 ng in a 10 ul ligation reaction or so). We do similar ligations routinely. You probably want to do ligations at 16C or below, because of the short length and AT composition of the PacI overhang.

Yes, for ligation I want to use 16C overnight. For DNA concentrations I was thinking to use more but I will try also with low concentration.
What about bacteria? Somebody told me that Stbl2 will be a good option because of the repetitions from the insert and the big size of the vector. What do you think? and by the way how much from the ligation do you use for transformation?

Thank a lot!

phage434 on Aug 26 2009, 11:59 PM said:

Hey,

Just try ligating the whole 3.3 kb insert. With vector size of 14 kb, avoid using agarose and just digest the vector, CIP it to avoid re-ligation of single cuts (if any), precipitate the digest, estimate DNA and ligate. I have worked with large constructs earlier and this strategy always worked for me.

Best,
TC

gbr on Aug 27 2009, 12:46 AM said:

Hi there!

Someone here with experience in cloning big plasmids?

I have a piece of 3.3kb to put into a 14kb vector. I tried to clone this piece before in a small plasmid (3kb) and only worked when I cut this piece in 2 : a 1kb fragment and a 2.3kb fragment. Initially this 2 pieces where separated by a neo casette but after removing it I was never able to work with the 3.3 kb - entire piece, I guess because I have some repetitive sequence there.

Anyway now I want to try a triple ligation :
Pac1::::::1kb piece::::::Bgl2
Bgl2:::::::2.3kb::::::::EcoR1
EcoR1:::::::vector::::::PacI (14kb)

Did anyone try something similar or has a recommandation for how to make this to work?

Thanks a lot!

I have 2 EcoR1 sites in the insert. If I cut with EcoR1 and Pac1 I will have again 3 fragments.

So for the triple ligation that I want to do I have to make to separate digestions: the additional EcoR1 it is in the 1kb fragment which I will cut out with Pac1 and BglII and I will make another digestion for getting the other fragment using the other site of EcoR1 and BglII.

In the vector EcoR1 and Pac1 are the only sites that I can use. Do you think that I have to do CIP for the 14 kb plasmid even if you have different ends ?

Thanks,
Gabriela

T C on Aug 27 2009, 08:56 AM said:

Hey,

Just try ligating the whole 3.3 kb insert. With vector size of 14 kb, avoid using agarose and just digest the vector, CIP it to avoid re-ligation of single cuts (if any), precipitate the digest, estimate DNA and ligate. I have worked with large constructs earlier and this strategy always worked for me.

Best,
TC

gbr on Aug 27 2009, 12:46 AM said:

Hi there!

Someone here with experience in cloning big plasmids?

I have a piece of 3.3kb to put into a 14kb vector. I tried to clone this piece before in a small plasmid (3kb) and only worked when I cut this piece in 2 : a 1kb fragment and a 2.3kb fragment. Initially this 2 pieces where separated by a neo casette but after removing it I was never able to work with the 3.3 kb - entire piece, I guess because I have some repetitive sequence there.

Anyway now I want to try a triple ligation :
Pac1::::::1kb piece::::::Bgl2
Bgl2:::::::2.3kb::::::::EcoR1
EcoR1:::::::vector::::::PacI (14kb)

Did anyone try something similar or has a recommandation for how to make this to work?

Thanks a lot!

Hey,

If I was you, I would insert the first EcoRI Pac I fragment, followed by inserting the EcoRI, EcoRI fragment (no problem with yr strategy, just that i like to keep things simple .

You don't need to CIP with different ends, I just do it as a precaution but not always.

The take home message is to avoid agarose and precipitating large fragments. Yr cloning would wrk in one go.

Best,
TC

gbr on Aug 27 2009, 02:07 PM said:

Thanks for ideas! I will keep them in mind for the next time.
Now it worked with the triple ligation and I am very happy , but I have the problem that I don't know how to transfect this 17kb plasmid in dopaminergic neurons primary cell culture. Do you have any tips?

Thanks a lot,
Gabriela

T C on Aug 28 2009, 04:18 PM said:

Hey,

If I was you, I would insert the first EcoRI Pac I fragment, followed by inserting the EcoRI, EcoRI fragment (no problem with yr strategy, just that i like to keep things simple .

You don't need to CIP with different ends, I just do it as a precaution but not always.

The take home message is to avoid agarose and precipitating large fragments. Yr cloning would wrk in one go.

Best,
TC

gbr on Aug 27 2009, 02:07 PM said:

I have 2 EcoR1 sites in the insert. If I cut with EcoR1 and Pac1 I will have again 3 fragments.

So for the triple ligation that I want to do I have to make to separate digestions: the additional EcoR1 it is in the 1kb fragment which I will cut out with Pac1 and BglII and I will make another digestion for getting the other fragment using the other site of EcoR1 and BglII.

In the vector EcoR1 and Pac1 are the only sites that I can use. Do you think that I have to do CIP for the 14 kb plasmid even if you have different ends ?

Thanks,
Gabriela

Hi there Gabriela

I need to set up triple ligation and I am not sure about the conditions. I was searching net for help so I run into this forum and your successful ligation. You was also in my position but at the end you made it!! Bravo!!! Can you pls provide me with the exect conditions that you used to make this thing to work? I know that its too much time since 2009 but I hope that I´ll get answer from you!!
Btw my name is Daniela

Thks a lot!!

All Best