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Team:HokkaidoU Japan/ProjectTest
From 2010.igem.org
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{{Template:HokkaidoU_Japan}} | {{Template:HokkaidoU_Japan}} | ||
=PCR based assembly protocol= | =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 | + | 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=== | ||
| + | ** Digestion Visualization Primers (DV Primers) | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | ===3. High accuracy 3 piece ligation=== | ||
| + | ** Quick and easy and accurate way to calculate reaction solution mixes | ||
Revision as of 11:35, 27 October 2010
since 13 Jul 2010
since 1 Aug 2010
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
- Digestion Visualization Primers (DV Primers)
3. High accuracy 3 piece ligation
- Quick and easy and accurate way to calculate reaction solution mixes
