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

Karmella's BioBrick Cloning

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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 hPCD + BL01 + V0120 hPCD + BL01 + pSB1A3 PCR V0120, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002) pSB1A3, Gib_BBS F (Gib0001) / Gib_BBP R (Gib0002) hPCD, BBP-hPCD fwd (BL9) / hPCD-BL01 rev (BL7) 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, ∞ 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: hPCD + BL01 + V0120 V0120 hPCD + BL01 + pSB1A3 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 hPCD + BL01 + V0120 hPCD + BL01 + pSB1A3 Check the sequences for BsmBI docking sites (CGTCTC) hPCD - none BL01: hPCD-mCherry-SP1AB - none V0120 - 5 sites pSB1A3 - none PCR V0120, gg_BBS F (gg0001) / gg_BBP R (gg0002) pSB1A3, gg_BBS F (gg0001) / gg_BBP R (gg0002) hPCD, gg_BBP-hPCD F (gg0003) / gg_hPCD-BL01 R (gg0004) 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, ∞ 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