Golden Gate Assembly protocol

Protocol to insert new promoter made from oligos into pSB1A2 with insert BBa_J100044

First, you need to dilute your assembled oligos. To do this, dilute them 200 fold.
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.
Now add 398 µL water to the 2 µL of cooled, annealed ologs. Mix this by stirring the tube with a yellow tip.

Get two new, small microfuge tubes designed to fit into the PCR machine.
One tube will be labeled "ligation", the other "plasmid only". Also put your initials on the tubes.


1 µL diluted oligos cooled overnight (use water instead for "plasmid only" tube)
9 µL ligations mixture provided by your instructor.
use the promoter DNA for the ligation tube but use only water for the plasmid only tube.

Ligation mixture contains:
1 µL (10-20 ng) plasmid containing part J100044
5 µL dH₂O
1 µL 10X Promega Ligase Buffer
1 µL 500 mM KOAc
0.5 µL Bsa I high fidelity (HF) restriction enzyme
0.5 µL T4 DNA Ligase
9 µL final volume

Turn on the PCR machine. Put both of your tubes into the machine.
Program it for the following cylces:
30 cycles of 37C for 1 minute/16C for 1 minute
1 cycle of 37C for 15 minutes
22C holding temperature
This DNA ligation is ready for transformation.

Transformations
You want to do 3 transformations:
a positive control
your experimental ligation
and your negative control ligation

Use all 10 µL of the ligations for a transformation.
You will want to perform a transformation positive control using K315008 which contains pLacI+RBS+RFP in plasmid pSB1A2.

Take one tube of 100 µL competent cells and put 30 µL of cells into each of three 1.5 mL tubes labeled appropriately for each of the three transformations listed above. Plate all three transformations on LB amp plates.

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