User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/05

01/05/13

• Golden gate: PCR parts, complete protocol for Brady's construct
• Gibson: PCR parts; waiting on BsmBI from NEB

Gibson Assembly

1. hPCD + BL01 + V0120
2. hPCD + BL01 + pSB1A3

PCR

1. V0120, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002)
2. pSB1A3, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002)
3. hPCD, BBP-hPCD fwd (BL9) / hPCD-BL01 rev (BL7)
4. BL01, hPCD-BL01 fwd (BL8) / BL01-BBS rev (BL10)

Reagent	Volume	Expected: G1. V0120 = 3200 G2. pSB1A3 = 2000 G3. hPCD = 186 G4. BL01 = 2520	10 μL/lane; 1% agarose; Ladder
DNA(plasmid)	0.5 μL
10 μM primer 1	1.0
10 μM primer 2	1.0
2x GoTaq mix	25.0
dH2O	22.5
	50 μL

• 95°C, 3 min.
• [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
• 72°C, 3 min.
• 4°C, ∞

Measure DNA Concentrations

Reaction set-up

• Use 0.2 - 0.5 pmol, per NEB's suggestion, [1]
• pmols = ng x 1000 / bp x 650 daltons; ng = (pmols x bp x 650 daltons) / 1000
• Total volume of DNA = 5.0 μL max

1. hPCD + BL01 + V0120

• vector V0120, 0.1 pmol = 208 ng = 1.9 μL (use 1.4 μL)
• 2:1 insert hPCD, 0.2 pmol = 24 ng = 0.7 μL
• 2:1 insert BL01, 0.2 pmol = 328 ng = 2.9 μL

2. hPCD + BL01 + V0120

• vector pSB1A3, 0.1 pmol = 130 ng = 1.3 μL
• 2:1 insert hPCD, 0.2 pmol = 24 ng = 0.7 μL (use 0.8 μL)
• 2:1 insert BL01, 0.2 pmol = 328 ng = 2.9 μL

Gibson reactions:

1. hPCD + BL01 + V0120
2. V0120
3. hPCD + BL01 + pSB1A3
4. pSB1A3

• Add each to one aliquot of 15.0 Gibson mix (Rene); total = 20.0 μL
• Thermal cycler: 50°C,60 min.; 4°C, ∞

Transformation

• (1) Transform: add 2.0 μL to 50 μL DH5α-Turbo; ice/5 min.; plate on warm 100 μg/mL amp. agar
• (2) NEB also suggests diluting 5.0 μL of the Gibson reaction into 15.0 μL dH₂O (20.0 total vol.), and using 2.0 μL diluted DNA
• Test both methods (8 plates total)
  • Plates 1-4, method 1
  • Plates 5-8, method 2

Golden Gate Assembly

• Try new protocol from Dave Savage

1. hPCD + BL01 + V0120
2. hPCD + BL01 + pSB1A3

• Check the sequences for BsmBI sites!

PCR

1. V0120, gg_BBS F (gg0001) / gg_BBP R (gg0002)
2. pSB1A3, gg_BBS F (gg0001) / gg_BBP R (gg0002)
3. hPCD, gg_BBP-hPCD F (gg0003) / gg_hPCD-BL01 R (gg0004)
4. BL01, gg_BL01 F (gg0004) / gg_BL01-BBS R (gg0005)

Reagent	Volume	Expected: gg1. V0120 = 3200 gg2. pSB1A3 = 2000 gg3. hPCD = 186 gg4. BL01 = 2520	10 μL/lane; 1% agarose; Ladder
DNA(plasmid)	0.5 μL
10 μM primer 1	1.0
10 μM primer 2	1.0
2x GoTaq mix	25.0
dH2O	22.5
	50 μL

• 95°C, 3 min.
• [95°C, 30 sec.; 57°C, 30 sec.; 72°C, 2 min.] x 30
• 72°C, 3 min.
• 4°C, ∞

Measure DNA Concentrations