three way ligation - three way ligation help (Jul/23/2007 )

hello all
I Am doing three way ligation with fast link DNA ligase from EPISCI .
the three fragment

1. The vector plasmid is PBS KS (-) 2.7Kb digested with bamHI and HindIII
2. Fragment I 3KB digested with hindIII and BglII
3. fragment II digested with BglII and BamHI.
(BglII and BamHi produces same type of sticky ends)
I tried many times bu tso far no sucess
any body please help me

thank u all
ALi

Normally, a 3-way ligation generates about 10% of the cfu of a 2-way ligation. In your case, fragment 3 can ligate to itself as well as the other 2 fragments in both orientations. Ligation with high DNA concentrations can lead to linear concatamers that contain multiple copies of fragment 3 in various orientations. All of these undesirable reactions are obscuring the correct 3-way ligation and reducing the frequency of obtaining it.

I assume that what you wnat is BamH I--Frag 1--HinD III/HinD III--Frag 2--Bgl II/Bgl II--Frag 3--BamH I/ or something similar since frag 3 can go in both orientations.
But another ligation product might be BamH I--Frag 1--HinD III/HinD III--Frag 2--Bgl II/Bgl II--Frag 3--BamH I/BamH I--Frag 3--Bgl II.
Another could be BamH I--Frag 1--HinD III/HinD III--Frag 2--Bgl II/Bgl II--Frag 2--Hind III.

While the correct product is not quite impossible to get, you will have to be extremely lucky. Most of the ligation products will not transform because they are linear.

I can think of one possible way to construct this plasmid:
BamH I--Frag 1--HinD III should be dephosphorylated with SAP so that contaminating single cut vector cannot religate.
HinD III--Frag 2--Bgl II should be prepared by first cutting with Bgl II and SAP (simultaneous), inactivate the SAP and do the second cut with Hind III (No SAP).
Bgl II--Frag 3--BamH I should be prepared by first cutting with BamH I and SAP (simultaneous), inactivate the SAP and do the second cut with Bgl II (No SAP).
The ligation products will be (BamH I)--Frag 1--(HinD III)/HinD III--Frag 2--(Bgl II)/Bgl II--Frag 3--(BamH I), or (BamH I)--Frag 3--Bgl II/Bgl II--Frag 3--(BamH) or (Bgl II)--Frag 2--Hind III/HinD III--Frag 2--(Bgl II), where a restriction site in parentheses indicates that it is dephosphorylated. But I think this eliminates all of the other ligation possibilities. Gel purify to obtain the desired 3-way product, kinase with cold ATP, and ligate the linear molecule into circular form for transformation.

It's a lot of work.

ditch the fast link DNA ligase. For multiway ligations, you can't use quick ligase buffers. Avoid any buffer with PEG in it.

I would conduct the multiway as an overnight ligation at 16 Celsius, using normal T4 ligase buffer (I use 0.5ul NEB T4 ligase (neat)... although I assume any normal ligase would do)

I assume you will be plating your transformant on an Xgal plate to conduct a blue/white colour test. In which case, you can easily remove empty vectors. You will however have to wary of BamHI/BglII fragment, if you a primer for that fragment, I would suggest that you use it.

Finally do remember that colony PCR is sensitive enough to pick up left over DNA insert from the ligation reaction sitting on your plate. So when doing colony PCR always amplify across junction, as that particular structure is unique to a ligated plasmid.

thank you very muc hfor your helpful replies
the construc will have double antibiotic resistance Amp and kanamycine.
Amp already in PBS KS and Kanamycine in the fragment 2. After ligation i am plating them on LB Amp plus kan plates. so far it give zero colonies.
So the selection for a correct plasmid will be easy if I get the ligation working and the correct contruct.

thanks
ali

the construct is shown in the following gif image

I am trying to change the BamHi site .
the options I have are
XbaI, NdeI. ScaI , HinFI

Impressive. Quite impressive.

I would go with either XbaI or NdeI.
However do note that XbaI is dam sensitive due to possible overlap with a dam recognition site. So when designing the XbaI site make sure you don't accidentally introduce a possible methylation site.

As for NdeI, it needs a larger then normal skirting of bp for the site to be cut efficiencyly. I would give that site a minimum of 8bps of skirting.

P.S: Just as a reminder, make sure that Kan gene's promoter and terminator has been clone as well. Not just the KanR ORF.