User:Karmella Haynes/Notebook/BioBrick cloning/2015/05/01

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05/01/15

  • Assembly: KAH87 (hPcTF ORF) into MV10



Assembly: KAH87 (hPcTF ORF) into MV10


  • Minipreps (5 mL cultures)
    • Sigma GenElute kit
    • Elute w/ 75 μL elution sln.
  1. KAH87
  2. MV10


  • Measure conc.'s of minipreps
Sample OD260 260/280 ng/μL
1. KAH87 (X/S) 0.180 1.923 179.8
2. MV10 (X) 0.371 1.926 370.5


  • Assemblies
  1. KAH87/MV10: KAH87/(X/S)/1053 + MV10/(X)/5197


  • Digests (Fermentas FD)
  1. KAH87, X/S
  2. MV10, X
Reagent Rxn 1 Rxn 2 Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20.0 15.0
10x buffer 3.0 3.0
XbaI 1.0 1.0
SpeI 1.0 ---
dH2O 5.0 10.0
  30 μL

--> 37°C/ ~30 min.


  • Gel purify
    • Sigma kit
    • Elute & back-elute w/ 25 μL elution sln.


  • Measure conc.'s
Sample OD260 260/280 ng/μL
1. KAH87 (X/S) 0.003 1.571 3.49
2. MV10 (X) 0.070 1.843 70.41


  • Dephosphorylation (Roche)
    • Dephos the MV10 (X) fragment
Reagent Volume
DNA (clean digest) 7.1 (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O 9.9
  20 μL

--> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


  • Ligations
    • 2:1 ratio calc.: 1053 (KAH87) / 5197 (MV10) * 2 * 50 = 20 ng KAH87
  1. KAH87 (X/S) + MV10 (X)
  2. MV10 (X dp), neg ctrl.
  1 2
Insert DNA 5.7 ---
Vector DNA 2.0 2.0
2x lgn buf (Roche) 8.7 8.7
T4 ligase (NEB) 1.0 1.0
dH2O --- 5.7
  17.4 μL 17.4 μL


RESULTS (5/01/15)

  • Success! Plate #1 has 50 colonies, neg ctrl has 1 colony
  • Pick 4 colonies (50% chance of fwd vs. reverse insertion)