How about 4 DNA fragments ligation and cloning - Cloning by 4 DNA fragments ligation and transformation (Feb/21/2007 )

Have any specialist succeeded in cloning of 4 DNA fragments ligation
( two adaptors parts plus two backbone parts of vector ,then transformation?)
& cloning . Any comment is greatly appreciated.

I happily do 5 way ligation
So 4 ways is certainly doable. But you need colony PCR and be prepared to screen a minimum of 100 colonies. In my hands the probability of success for a 4 ways is about 1:20 to 1:25 colonies.

However I am very concern about the use of adaptors/linkers. Aside from small linkers, I have not had a good experience with them. There is a problem (perhaps with my linker annealing protocol) because I constantly find mistakes in my linkers. error rate ranges from 1:3 to 7:8 of all sequenced plasmids. Yes, I have a poor success rate here. Thus I dislike using large linkers.

May I know how large are the linker you intend to use? If you are flanking a DNA segment with linkers, would it not be possible to simply PCR amplify said DNA segment with primers containing the sequences you had wished to add by linkers? This looks much easier.

lucky guy

i am even afraid of 2 way ligation..............
day by day its been e phobia to me

Don't be. With proper design and non compatible ends, multiways are not difficult.

Give it a try. Design your primers such that in the worst case senario you can fall back onto a 2way ligation. You will soon come to regret ever doing anything by 2 way ligations.

thanks ot encourage me, i really appreciate it

I happily do 5 way ligation
So 4 ways is certainly doable. But you need colony PCR and be prepared to screen a minimum of 100 colonies. In my hands the probability of success for a 4 ways is about 1:20 to 1:25 colonies.

However I am very concern about the use of adaptors/linkers. Aside from small linkers, I have not had a good experience with them. There is a problem (perhaps with my linker annealing protocol) because I constantly find mistakes in my linkers. error rate ranges from 1:3 to 7:8 of all sequenced plasmids. Yes, I have a poor success rate here. Thus I dislike using large linkers.

May I know how large are the linker you intend to use? If you are flanking a DNA segment with linkers, would it not be possible to simply PCR amplify said DNA segment with primers containing the sequences you had wished to add by linkers? This looks much easier.

I happily do 5 way ligation
So 4 ways is certainly doable. But you need colony PCR and be prepared to screen a minimum of 100 colonies. In my hands the probability of success for a 4 ways is about 1:20 to 1:25 colonies.