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PCR Cloning
bioforum - 24 Jul 2013
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Couple points. You must use a buffer that contains a detergent such as Taq buffer. Most pfu buffers do not have a detergent and therefor will not efficiently break open the bacterial cells. Also, be aware that false positives are a potential downfall of pcr screening. When you add the ligation mixture to the competent cells during transformation, there is free insert DNA present. That DNA gets spread onto the LB plate and can be picked up by your tip when picking a colony. Always do a second diagnostic test once you have purified the plasmid.