Ligating multiple DNA fragments - (Sep/15/2006 )

Hi there,

I am having some trouble with a ligation procedure. I am attempting to piece together three DNA fragments. I have a PCR product that I'd like to put in between two regions for homologous recombination down the road. Has anyone ever tried ligating more than two fragments with any success? These fragments are not anchored in a vector.

Thanks for your help!

-Cheamps-

Hi， I guess the complexity of procedure also depends on the restriction enzymes you are planning to use. you can either ligase two fragments together first and then ligate the third fragment, alternatively, you can put 3 together and then isolate the product you want.

-angelaileen-

QUOTE (Cheamps @ Sep 15 2006, 10:02 PM)

Yes, I have done 5 way ligation (4 inserts into 1 vector), with a colour testable vector. EVerything was thrown into one ligation mix.

If the ends of the fragments are cut (with restriction enzymes that make it) such that the fragments can ligate together in only one manner.

Furthermore, if your vector is dephosphrylated and colour testable or contain some form of positive selection. ie something to discriminate empty vectors verse vectors carrying inserts

You should be fine.

Be willing to screen at leat 96 colonies (with colony PCR). The process is very stocastic and luck prone... you may get nothing for the first 40 colonies, and then 3 in the next 10.

QUOTE (angelaileen @ Sep 15 2006, 11:53 PM)

you can either ligase two fragments together first and then ligate the third fragment, alternatively, you can put 3 together and then isolate the product you want.

Is also a good idea. I have considered using this approach for ligations more then 5 ways. But I have yet to try... mainly because I have yet to need to do a 6 way. (But knowing my boss, it maybe soon... as even 5 ways is too slow. sigh)

-perneseblue-

We do three-way ligations into a suicide vector for allelic replacements all the time...

-HomeBrew-

Thanks, everyone. I am using XbaI and PacI to create sticky ends so my insert can ligate in between the two flanking regions. I then ran the digests out on a gel, extracted the fragments I wanted, and tried ligating in one mixture (rather than a sequential ligation). I can't seem to get the construct I want. I get many dimers or trimers of the flanking regions, but haven't had much luck getting the flanking regions actually flanking my insert (GFP).

I am considering running a PCR on a gel extraction of the area my construct should be in, to see if my construct is there but simply in a low quantity as compared to the rest, but there has to be a more efficient method. What do you think?

-Cheamps-