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SLIC Cloning Failure

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#1 chaichontat



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Posted 18 November 2015 - 12:13 AM

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.

Edited by chaichontat, 18 November 2015 - 02:02 AM.

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