Latest revision as of 11:48, 27 October 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

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

@@ Line 1: / Line 1: @@
 {{Template:HokkaidoU_Japan}}
-=Project Abstract=
+=PCR based assembly protocol=
-<p>
+&nbsp;&nbsp;&nbsp;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.
- Our project is on Type III Secretion Apparatus which is one of the most amazing biological devices. It can pass a whole protein molecule from a bacterial cell to a target eukaryotic cell. This apparatus which looks like a syringe is an organelle of pathogenic gram-negative
-bacterium such as Salmonella and Yersinia. We are aiming at making this device available for ''E. coli''. Because it will not involve usage of pathogenic strains, it will be safer to use. To transfer T3SS functionally from Salmonella to ''E. coli'' it is essential to integrate at least 40kb of DNA fragment coding more than 20 proteins. So we will make suggestions about how to optimize ''E. coli'' transformation method for large size DNA fragments. Also we will show how to construct protein for secretion and how to measure if it is really secreted using GFP.
-</p>
+==Key Requirements for PCR Based Assembly Protocol==
+===1. Reliable proofreading PCR enzymes===
+&nbsp;&nbsp;&nbsp;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.
+&nbsp;&nbsp;&nbsp;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.
-==Construction of GFP injector by ''E. coli''==
+===2. Visualization of DNA Complete Digestion===
+&nbsp;&nbsp;&nbsp;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.
-==Methods for Infection Analysis==
+===3. High accuracy 3 piece ligation===
+** Quick and easy and accurate way to calculate reaction solution mixes
-==Results==
-==Discussion==
-==Acknowledgements==
-==References==

Team:HokkaidoU Japan/ProjectTest

From 2010.igem.org