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

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01/05/13

  • Gibson: PCR parts, complete protocol for Brady's construct
  • Golden gate: 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
PCR gel
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, ∞


Purification

  • Cleaned remaining 40 μL of PCR using the Zymo DNA Clean & Concentrator kit
  • Eluted samples with 20 μL dH2O


DNA Concentrations

Sample OD 260 260/ 280 ng/μL
G1 V0120 0.108 1.801 108.375
G2 pSB1A3 0.101 1.849 100.553
G3 hPCD 0.034 1.876 33.586
G4 BL01 0.113 1.828 112.79


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
Reagent 1 2 3 4
Vector 1.4 1.4 1.3 1.3
Ins 1 0.7 --- 0.8 ---
Ins 2 2.9 --- 2.9 ---
dH2O --- 3.6 --- 3.7
  5.0 μL 5.0 μL 5.0 μL 5.0 μL
  • 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 40 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, [2]
  • 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 docking sites (CGTCTC)
    • hPCD - none
    • BL01: hPCD-mCherry-SP1AB - none
    • V0120 - 5 sites
    • pSB1A3 - none


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
PCR gel
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, ∞


Purification

  • Cleaned remaining 40 μL of PCR using the Zymo DNA Clean & Concentrator kit
  • Eluted samples with 20 μL dH2O


DNA Concentrations

Sample OD 260 260/ 280 ng/μL
gg1 V0120 0.099 1.834 98.549
gg2 pSB1A3 0.068 1.758 67.725
gg3 hPCD 0.022 1.72 22.485
gg4 BL01 0.056 1.707 55.883




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