Team:Johns Hopkins-BAG/Protocols

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[http://baderlab.bme.jhu.edu:8888/moodle18/ Moodle] [http://baderlab.bme.jhu.edu/syntheticyeast/wiki/index.php/Main_Page Synthetic Yeast Wiki] Protocols References Acknowledgements Contact

Contents 1 Protocols 1.1 The Ligase Chain Reaction Protocol 1.1.1 Introduction 1.1.2 Theory 1.1.3 Part 2

Protocols

The Ligase Chain Reaction Protocol

Introduction

The Ligase Chain Reaction Protocol (LCR) is designed to replace and improve the current Templateless Chain Reaction Protocol (TPCR). The LCR is thought to have many distinct advantages: 1.) Accuracy of BB sequence should be increased (theoretically). 2.) The actual construction of the BB should be considerably easier thanks to the overlapping feature of the oligonucleotides. 3.) Money should be saved throughout the entire workflow because the LCR only needs to be run once to produce workable results. In order to make a working protocol we must first subject the given protocol to stress tests to test for robustness. The second part of our experiments should be to test known working samples of the LCR trials in order to prove that the experimental procedure works correctly. (We did this because our oligonucleotides were built incorrectly) We used samples from Jennifer Tullman’s batch of 3L.3_23.A1 BB. The third part of our testing will be to assemble the failed BB’s from the Intersession 2009 work that Mary and I worked on.

Theory

The theoretical background for the efficiency and accuracy of the LCR is based on the un-gapped oligonucleotides and manner of construction of the BB.

Step 1
Build un-gapped oligonucleotides that are around 60bp each
Step 2a
Mix the oligonucleotides together.
Step 2b
Phosphorylate the 5’ ends of the oligonucleotides so that the Taq DNA Ligase (Thermostable) can work on ligating the spaces
<PICTURE>
Step 3 Use a chained reaction to allow the overlapping pieces to anneal and then use the ligase to join the DNA backbone.
<PICTURE>
This is the step where the efficiency and the accuracy are most obvious. The oligonucleotides have a very high specificity for their complementary strand. This is thanks to the ~30bp stretch of complementary DNA. There is also a very low possibility for loxP sites since a palindrome sequence is much harder to find as the stretch of complementary sites for annealing grow longer.

Step 4
Every step thereafter is the same as the old BAG protocol.

Part 2