View source for Golden Gate Assembly protocol

'''Protocol to insert new promoter made from oligos into pSB1A2 with insert [http://partsregistry.org/Part:BBa_J100028 BBa_J100028]'''<br>  <br>

First, you need to dilute your assembled oligos. To do this, dilute them 200 fold. <br>
The easiest way to do this is to remove 2 µL from the 20 µL overnight mixture and place this 2 µL into a new, 1.5 mL microfuge tube. <br>
Now add 398 µL water to the 2 µL of cooled, annealed ologs.  Mix this by stirring the tube with a yellow tip. <br> <br>

Get '''two''' new, small microfuge tubes designed to fit into the PCR machine.  <br>
One tube will be labeled "ligation", the other "plasmid only". Also put your initials on the tubes. <br> <br> <br>
1 µL diluted oligos cooled overnight (''use water instead for "plasmid only" tube'')<br> 
9 µL ligations mixture provided by your instructor. <br> 
use the promoter DNA for the ligation tube but use only water for the plasmid only tube. <br> <br> 

Ligation mixture contains:<br>
1 µL (10-20 ng) plasmid containing [http://partsregistry.org/Part:BBa_J100028 part J100028]<br> 
5 µL dH<sub>2</sub>O <br> 
1 µL 10X Promega Ligase Buffer<br> 
1 µL 500 mM NaCl<br> 
0.5 µL Bsa I high fidelity (HF) restriction enzyme<br> 
<u>0.5 µL T4 DNA Ligase</u><br> 
9 µL final volume <br> <br> 

Turn on the PCR machine. Put '''both''' of your tubes into the machine. <br> 
Program it for the following cylces: <br> 
30 cycles of 37C for 1 minute/16C for 1 minute <br> 
1 cycle of 37C for 5 minutes <br>
22C holding temperature <br> 
This DNA ligation is ready for transformation. <br> <br> 

'''Transformations''' <br> 
Use all 10 µL of the ligations for a [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html transformation]. <br> 
You will also want to perform a transformation positive control using [http://partsregistry.org/Part:BBa_K315008 K315008] which contains pLacI+RBS+RFP in plasmid pSB1A2. <br>  <br>

Take one tube of 100 µL competent cells and use 30 µL of cells for each of the three [http://www.bio.davidson.edu/courses/Molbio/Protocols/Zippy_Transformation.html transformations] listed above. Plate all three transformations on LB amp plates.<br>  <br> 


This protocols was developed at MWSU by Dr. Todd Eckdahl, and modified at Davidson College by students in Biology 111 and Romina Clemente.