User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/06

01/06/13

• Gibson Assembly: pick colonies & check via colony PCR
• Media: Make LB agar +amp
• Gibson Assembly: redo transformation with more DNA

Gibson Assembly results

• Plates from 01/05/2013

• hPCD + BL01 + V0120, 2μL 1x Gibson: no colonies for assembly (1) or no-insert control (2)
• hPCD + BL01 + pSB1A3, 2μL 1x Gibson: 1 colony for assembly (3), none on control (4)
• hPCD + BL01 + V0120, 2μL 1/4x Gibson: 2 colonies for assembly (5), 1 on control (6)
• hPCD + BL01 + pSB1A3, 2μL 1/4x Gibson: 1 colony for assembly (7), 2 on control (8)

• Pick and streak colonies from plates 3, 5, 7
• Use same pipette tip for colony PCR

Reagent	Volume	5x mix	30 μL/lane, 1% agarose; Ladder
Colony	---	---
10 μM primer BL9	1.0	5.0
10 μM primer BL10	1.0	5.0
2x GoTaq	12.5	62.5
dH2O	10.5	52.5
	25.0 μL

PCR

• 95°C, 3 min.
• [95°C, 30 sec.; 95°C, 30 sec.; 72°C, 1 min.] x35
• 72°C, 3 min.
• 4°C, ∞

Conclusion: looks like primer dimer. Re-do the transformation with 3x more DNA (from yesterday, stored at -20°C)

Transformation

1. hPCD + BL01 + V0120, 6.0 μL 1/4x Gibson
2. V0120, 6.0 μL 1/4x Gibson
3. hPCD + BL01 + pSB1A3, 6.0 μL 1/4x Gibson
4. pSB1A3, 6.0 μL 1/4x Gibson

• Add DNA to 50 μL DH5α-Turbo; ice/ 10 min.
• Plate on 100 μg/mL amp.