An expert guide to PCR troubleshooting. The online guide covers problems with the template DNA, inadequate dNTPs, primer concentration, Taq concentration, Mg concentration, and KCl concentration. Most PCR problems are caused by one or other of these variables.

Find out the theory and possible solutions to these problems at PCR troubleshooting.

Labels: trouble shooting, Troubleshooting

Zuo and Rabie describe a new one-step "Quick Assemble" method of precisely and simultaneously joining multiple DNA fragments followed by intramolecular ligation. This technique of plasmid assembly and circularization can be used to construct new plasmids with any promoter, resistance gene marker, restriction site, or tag.

The new 2-day PCR-based method can be used to generate both circularized and linear final assembled products. The method makes the synthesis and assembly of large molecules in a quick and efficient way, while circumventing the use of DNA ligases. The ligase-free method also offers an additional improvement over the combination of long PCR and overlap extension PCR, and is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as multiple site-direct mutagenesis and whole-DNA library gene shuffling.

from Zuo and Rabie in Curr. Issues Mol. Biol. 12: 11-16

Further reading: Assembly of DNA fragments into circular constructs

Labels: cloning, PCR cloning