User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/10

01/10/13

• Violacein: successful growth; re-streak
• Golden gate/ transformation: transform DH5α-Turbo with gg assemblies from 1/09/13
• Golden gate trial 2: new PCR primers

Violacein

• Plate 1: Kan plasmid
• Plate 2: Amp plasmid (violacein exp.)

Golden gate trial 1

• Add total 10.0 μL run to 50 μL DH5α-Turbo; incubate on ice 10 min.; plate on amp agar
• Colonies: 7 on assembly plate, 9 on vector-only control. Pick 4 and seed 2 mL liquid cultures.

Golden gate trial 2

• Re-designed primers to include "handle" preceding the BsmBI sites and a spacer between BsmBI and a 4 bp overlap region

Reagent	Volume	Expected: 1. pSB1A3 = ~2000 2. hPCD = 186 3. BL01 = 2520	10 μL/lane; 1% agarose; Ladder
DNA(plasmid)	0.2 μL
primer 1	1.0
primer 2	1.0
2x GoTaq	25.0
dH2O	22.8
	50.0 μL

PCR

• 95°C, 3 min.
• [95°C, 3 min.; 95°C, 3 min.; 95°C, 3 min.] x35
• 72°C, 3 min.
• 4°C, ∞

Purification

• Clean with Zymo DNA c&c; elute w/ 20 μL dH₂O
• Measure [DNA]

Dilutions

• Golden Gate Reactions

1. hPCD + BL01 + pSB1A3
2. pSB1A3

Thermal cycler

• [45°C, 2 min.; 16°C, 5 min.] x25
• 60°C, 20 min.
• 80°C, 20 min.