Double ligation - What about cloning two fragments in the same reaction? (Dec/19/2007 )

Hi all,

I searched the forum hoping to see some information on double ligation but I couldn't find any post on this subject. Is there anything I should know before trying to ligate 2 different fragments in a single vector and in a single reaction. Let's say the fragments are designed as follow: fragment 1= 5'-XbaI-XXXX...XXXX-EcoRI-3', fragment 2= 5'-EcoRI-xxxx...xxxx-BamHI-3' and that the vector has both XbaI and BamHI sites. Chances of getting colonies producing the vector with both fragments should be high since the vector will be digested with XbaI and BamHI, making it impossible for only one of the 2 fragments to insert alone, right? What's your experience with such experiments?

And what about a triple ligation using the same design?

KenQ

This should "just work" if you have high quality fragments. Same with triple insert ligation.

And 4 way ligation. (3 inserts 1 vector)

And 5 way ligation. (4 inserts 1 vector)

Here are a few don't that you should watch out for....
Don't over expose your DNA fragments to UV. Time to excise the gel slabs that contains your DNA fragments should be counted in seconds. Exposure to UV severely reduces the DNA's tranformabillity. If you are getting sunburns from the UV transluminator... that is not a good thing....

Vector dephosphorylation is a tricky thing. Without enough experience, dephosphorylation can often times it be more hindrance than help. Since your vector has noncompetible ends, dephosphorylation is not required. Rule of thumb for dephosphorylation is 1unit CIP dephosphorylates 1pmol of DNA in 60mins at 37 Celsius within a 50ul volume in NEB buffer 3.

Personally I find it easier to work with large quantities of DNA. I cut 15ug of DNA. Very wastful by any standard, but I have never lost my DNA, or worried about the Qiagen column eating my DNA, or needed to repeat a digest. There is so much DNA that you can afford to lose DNA at every step.

DNA which is too concentrated will not cut. And less is more with regards to usage of restriction enzymes. The total volume of restriction enzymes in use must not exceed 5% of the digestion mixture's volume. The enzymes come in a protective buffer containing glycerol, unfortunately glycerol also inhibits the enzymes activity. So too much enzyme results in too much glycerol which means oddly less to no enzymatic activity.

Personally I gel purify all my vector and insert fragments, even when gel purification is not abolutely required. There is this phenomena of denatured plasmid. Denature plasmid is formed from over exposure to alkaline conditions during the alkaline lysis step. Denatured plasmid is somehow misfolded and becomes resistent to restriction enzymes however this DNA can still transform well enough. As can be expected this can be a sourse of problems especially if the DNA prep the vector came from was poorly conducted.

Quick ligase (buffer) is okay for two way ligation (1 insert; 1 vector). However for multiway ligations, normal T4 ligase buffer is required. Multiligation events are severely reduced when using quick ligase buffer.

Finally always check you work first before doing it. Use something like Vector NTI to keep a record of the plasmids that you have made and too make sure you don't have any unexpected restriction sites 'suddenly' appearing.

Thanks for the tips, pernesblue.

What's the concentration of DNA you use for the ligation? You start with 15ug so you must end up with a lot of DNA for your ligation, isn't it? And what's the insert:fragment ratio you use, is it something like 3:3:1 or 10:10:1?

Usually a ligation would use 100ng to 400ng of vector.

The molecule ratio of vector to insert(s) I usually use is 1:1:1.

Thus both the size of the DNA fragment and the concentration of DNA is considered.
I use the ratio of molecules, not the ratio of mass.

Instead of ligation, you could try homologous recombination. There are kits out there that allow you to insert multiple genes into your vector all in one reaction. I have found the cloning kit by BPS Bioscience to be the best because of it's efficiency and also becuase it doesn't require entry vectors like some others (TA cloning, topos, gateway, etc). I use the kit almost everday and I swear by it. I no longer need my good luck charm everytime I do cloning.