I use the In-Fusion system, this is a ligation independent system. Their site provides much of the information you will need to run any experiment you may need. They also have a tool that will generate primers with the proper overhangs to make the modification you desire. I have included the small flyer from their website that gives a schematic of how the system works. 

Dear Akshay

You can find some information about LIC protocol in the attached file.

Personally, for simple cloning, i prefer the PIPE cloning methods (you can find information about it in my profile) respect than the LIC. 

good luck

Manuele

You can also use competent cells that can ligate your cut vector and insert in vivo.

yes I agree with Manuele Martinelli 

I use the In-Fusion system, this is a ligation independent system. Their site provides much of the information you will need to run any experiment you may need. They also have a tool that will generate primers with the proper overhangs to make the modification you desire. I have included the small flyer from their website that gives a schematic of how the system works. 

Thank you @Manuele. That document is so helpful.

but still I cant understand primer designing for this method.

you need to read the specific requirement of the primer design related to your vector. for example using pET 101 D-TOPO vector, the vector contains overhang sequence of GTGG at the 3' end so when designing the primer, they strictly recommend that you should include CACC before the start codon (ATG) of your sequence. ie it should looks like this CACCATG........... The CACC is virtually another overhang from your primer. it i s the one that will hybridize with your GTGG from the vector, hence no need of the ligase.

Use recombineering..( DY380) was the strain i used. Its very efficient  and is LIC

Dear Akshay

you can find an example of primer desing for lic in the following link: 

www.embl.de/pepcore/pepcore_services/cloning/cloning_methods/lic/

i wish that i can help you to solve your doubts.

ciao

manuele

Hi Akshay,

How about trying SLiCE (http://www.cc.kyoto-su.ac.jp/~motohas/motohashi_lab/oyakudachi_others_SLiCE.html).

Though the web site is written in Japanese, you may understand figures.  And you can access to the papers that described this technique.  This is efficient, and less expensive than other system.  Recently, I constructed many plasmids with this method.

shigeki

Hi Ashkay-

Are you interested in cloning one insert into a vector? Or multiple inserts simultanously? If this is the first time, i would suggest you try with 1 insert. I recommend using In-fusion cloning like Ryan suggested above. 

Here is the basic workflow. Check this link:

http://www.clontech.com/US/Products/Cloning_and_Competent_Cells/Cloning_Resources/Online_In-Fusion_Tools

1. Choose "cloning"

2. Copy your vector sequence in to the box

3. Choose how you will linearize your vector (I recommend 2 restriction enzymes)

4. Input your insert sequence

Click "design primers"

Each primer will have 15bp overlap (complementary to the ends of linearized vector sequence) + additional bases to amplify from your insert sequence. Place the order for these primers.

When you receive the primers, design PCR reaction to amplify your fragment. Use PCR enzyme (CloneAmp) included in the In-fusion kit to amplify your insert. Gel purify.

Linearize your vector (make sure you have complete digestion if using RE) and then gel purify. 

Set up your In-fusion cloning reaction (combine together In-fusion enzyme premix with linearized vector + purified insert). The proprietary enzyme mix contains a 3'-5' exonuclease which then chews back the complementary ends of the vector/insert. The nicks are then repaired in E.coli.

Incubate the reaction for 15 minutes at 50C, then place on ice

Transform the Stellar competent cells (included in the kit) with 2.5uL of the In-fusion reaction mixture 

Thank you @Liz , but this website doesn't have BsaI RE option which I am going to use for my experiment.

Hi Akshay- 

The website does include the isoschizomers of Bsa1, Bso31I or Eco31I. Check out the NEB link below. You can choose either one of these options in place of Bsa1 in the program.

https://www.neb.com/tools-and-resources/interactive-tools/enzyme-finder?searchType=2&name={767F0D0D-C32A-4CA0-8436-FFB710F4CAC7}

Hi Akshay,

The following web site is quite useful. Though the principle is described in Japanese, basic protocol is written in English. This method, called SLiCE, is cheap and efficient.

http://www.cc.kyoto-su.ac.jp/~motohas/motohashi_lab/oyakudachi_others_SLiCE.html