Golden Gate Assembly with Pclone Red

Using Synthetic Biology and pClone Red for Authentic Research on Promoter Function: Introductory Biology (identifying new promoters)

A. Malcolm Campbell and Todd T. Eckdahl

Golden Gate Assembly with pClone Red

On the day when Golden Gate Assembly is to be performed, preparea master mix for the students that includesBsaI, T4 DNA Ligase, and Ligase Buffer.

Directions for 8 Lab Groups, 4 in the morning and 4 in the afternoon

Anytime before lab, store -20° C
1) Aliquot J119137 (receiving plasmid) to 73 ng/ µL (make at least 21 µL volume)

2) Aliquot J04450 (+ control) to 50 ng/ µL (make at least 10 µL volume)

3) Put 1 µL of J04450 into 9 tubes so that every group gets one tube (label with + (plus) on lid for positive control). This tube will be used for the RFP positive control.

4) GGA Mixture contains these ingredients that can be made in advance (stored at -20° C):

10 µL J119137 (73 ng/µL)
60 µL water
10 µL 10X ligation buffer (included with Promega#M1804)
10 µL 500 mM potassium acetate (make stock solution at your institution)

Make two tubes with the volumes listed above. Label one tube AM and one tube PM for the two lab sections. Each tube is enough for 4 groups, with each group using 2 tubes, plus a spare.

Day of the lab
5) Just before the lab, thaw one of the GGA Mixture tubes and add the following:

5 µL BsaI HF (New England BioLabs, #R3535L)
5 µL T4 DNA ligase(New England BioLabs, #M0202S)

6) For each lab section, mix the ingredients and aliquot 9 µL into 4 tubes (that fits your thermocycler machine) so that each group gets two tubes, plus one spare. Label one “P” for (promoter) and one “—” (minus for negative control). Store GGA mix on ice.

7) Students will pipet 1 µL of their cooled and dilutedoligos into the P tube and 1 µL of water into the “—”(minus) tube. These tubes are placed into the thermocycler to perform GGA. The thermal profile is 20 cycles of (1 minute at 37° C and 1 minute at 16° C), followed by one cycle of 15 minutes at 37° C, and a final hold at room temp. The + (plus) tube is only used for transformation, so no GGAis required.

Students performed GGA reactions in a total volume of 10 µL containing

6 µL H2O
1 µL 10X T4 DNA Ligase Buffer (includedwith order of ligase as 300mM Tris-HCl (pH 7.8 at 25°C), 100 mM MgCl2, 100 mM DTT and 10 mM ATP)
1 µL of 40 nMpCloneRed
1 µL of 40 nM annealed promoter oligonucleotides
0.5 µL HF (high fidelity; New England BioLabs) BsaI (10 units)
0.5 µL T4 DNA Ligase (New England BioLabs; 50 Weiss units).

For each lab group, we prepared a 8 µL master mix containing all of the components except the annealed oligonucleotides and the pClone destination plasmid before class and stored it in the refrigerator until needed later in the day. Students mixed the master mix, annealed oligos, and pClone destination plasmid. They placed their GGA reactions in a thermal cycler set for 20 cycles of 1 minute at 37° C followed by 1 minute at 16°C. The 37°C temperature favors digestion by BsaI-HF while the 16°C favors ligation by T4 DNA ligase. GGA terminates with a 15 minute incubation at 37° C to cleave any remaining BsaI sites. Students also performed a negative control GGA in which the annealedoligos are replaced by water but all other components are kept the same.