Golden Gate Assembly protocol

To insert oligos into pSB1A2 with insert BBa_J100028
1 µL assembled oligos cooled overnight
1 µL (10 ng) pSB1A2+J100028
5 µL dH2O
1 µL 10X Promega Ligase Buffer
1 µL 500 mM NaCl
0.5 µL Bsa I restriction enzyme
0.5 µL T4 DNA Ligase
10 µL final volume

Use all 10 µL for a transformation.
You will want to do a negative control where you leave out the assembled oligos and add one more microliter of water.
You will also want to perform a positive control of K315008 which contains pLacI+RBS+RFP in plasmid pSB1A2.

Take one tube of 100 µL competent cells and use 30 µL of cells for each of the three transformations listed above. Plate all three transformations on LB amp plates.

This protocols was developed at MWSU by Todd Eckdahl, and modified at Davidson College.