SLIC Cloning Failure - (Nov/18/2015 )

Hi all,

I have been trying to clone two gene fragments into a vector by the SLIC method. However, there's no significant difference in the number of colonies in the negative and positive plate. In some scenario, the positive plate had even less colonies than the negative plate. The backbone was XbaI-linearized pUC19, and all fragments had 20 bp homology with each other with a molar ratio of 1:2:2 to  a final concentration of 7 ng/ul.

I tried both protocols I found, the first being Li et al. (2007) Nature Methods (incubate the mix for 30 min then add dCTP and mix everything together). The second protocol is Jeong et al. (2012) where all fragments were mixed at once, incubated for 2.5 min, and put on ice for 10 min. After both protocols, 5ul of the mix was transformed into 100ul of DH5a cells.

What am I missing here? Any help would be appreciated.

I tried these methods before. They work OK.

However, now I use commercial ones like In-fusion (Clontech) and Gibson assembly (NEB) and they work much better than using the above methods.

Is the xbaI working correctly?

Are you sure you have correct homology also for the vector plasmid?

5' end of fragment 1 must have homology with 3'  end of your vector.

3' end of fragment 1 must have homology with 5' end of fragment 2

3'' end of fragment 2 must have homology with 5' end of vector.

The usual problem is incomplete cutting of the parent plasmid. It is very hard to drive the XbaI cutting to completion, and even if complete, some religation may occur when repairs happen inside cells. Better is to use vanishingly small amounts of plasmid template and PCR to amplify linear DNA  from the plasmid. You can cut the parent plasmid with DpnI as well.