PCR based assembly protocol

   In standard protocol, DNA fragment is amplified via transformation and miniprep. But this takes too long time you know. Moreover, minipreped plasmid is impure in most cases. So we did PCR based assembly in this summer, and encounterd some key requirements.

Key Requirements for PCR Based Assembly Protocol

1. Reliable proofreading PCR enzymes

   We used KOD Plus NEO currently popular only in japan. This enzyme has 80 times more reliable proofreading activity than Taq polymerase. It is particularly useful when we amplify the BioBricks from Distribution Kit.    In order to amplify the DNA after initial transformation, we have to extract plasmid via miniprep. But, we thought that miniprep can be skipped altogether by doing single colony PCR and extracting amplified fragments.

2. Visualization of DNA Complete Digestion

   When you amplify parts by PCR using primers which anneal to prefix and suffix sites, it is uncertain if digestion was successful. Because you can’t see any differences before and after when electrophoresis is performed. Then, Digestion Visualization primer set (DV primer set) removes this ambiguity. This primer set anneals 100 bp upstream of prefix and 200 bp downstream of suffix site.

3. High accuracy 3 piece ligation

• • Quick and easy and accurate way to calculate reaction solution mixes