Which one is more efficient?
When can infusion be more indicated?
Hi, I have been using the both strategies, RE and In-Fusion Clontech, and for long inserts (I am cloning 8000bp insert to 13000bp vector) In-Fusion is working much more better, just you have to design the primers but in Clontech web site there is a facility for doing that. Good luck!
Hi, I have been using the both strategies, RE and In-Fusion Clontech, and for long inserts (I am cloning 8000bp insert to 13000bp vector) In-Fusion is working much more better, just you have to design the primers but in Clontech web site there is a facility for doing that. Good luck!
Hi Antonio, agreed with Yordan, In-Phusion is much more efficient and quicker when cloning large fragments (let's say over 3000 bp) or if you need to join multiple pieces, which you can do in one cloning step. It also frees you from RE on the insert, you only need to open up the vector, and for this any RE is good.
If you want a cheaper version of the In-Fusion, you can go with NEB's Gibson Assembly, which uses similar principles, although the enzymes are different and the reaction times are a bit longer to equal In-Fusion efficiency.
For small fragments, small cDNAs, probes I still go with the classical RE system, I would say for now it's about 50-70% cheaper if you know your way and clone in one shot.
In-Fusion is also quite convenient when you don't want to depend on restriction sites, e.g. if you want to perform seamless cloning, or when you want to delete or exchange a specific region of a gene. A very efficient protocol is described in Benoit et al, 2006. Prot. Expr. and Purif.45, 66-71
I am trying to do a lot of cloning using In fusion from Takara. It had worked previously. But now I am simply not getting any colonies.
1. i designed primers using the primer designer tool in their site (only added a nucleotide to adjust frame)
2. I am purifying digested vector from gel with good yield (40/50 ng/ul).
3. I treat the PCR product with clonase enhancer but I did try purifying them from gel too with the yield of around 20-25 ng/ul. I perform in fusion reaction at 50 oc for 30 min.
4. I have used home made DH5alpha comptetent cells and Stellar competent cells too..But no colonies.
5. I am cloninng relatively large fragments into a large vectors usually (1.7 kbp into 8 kbp vectors).
6. Any suggestions?
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