Haynes Lab:Notebook/Engineering PC-TFs/2013/10/19

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October 19, 2013

Ligation of CMV + MV9

Part Concentration
CMV (insert) 15.535 ng/μL
MV9 (vector) 20.838 ng/μL

Calculations

ng insert = bp insert/bp vector * 2 * 50
ng insert = 588/4611 * 2 *50 = 12.75211 ng

μL insert = ng insert/concentration insert
μL insert = 12.75211/15.535 = .82μL

μL vector = 50 ng/concentration vector
μL vector = 50/20.838 = 2.4 μL

1 Control
DNA Insert 0.8 0
DNA Vector (MV9) 2.4 2.4
T4 Ligase 1 1
Lign Buffer (2x) 5 5
dH2O 0.8 1.6
Total 10 µL 10 µL


Bacterial Transformation of CMV Promoter + MV9

  • Warm 100 μg/mL Amp agar plate at 37°C
  • Thaw fresh tube of DH5α Turbo cells on ice
  • Add 30 μL of DH5α Turbo Cells to DNA + dH2O.
    • This was also done for transformation of part KAH126
  • Incubate cells + DNA on ice for 5 minutes.
  • Label pre-warmed plate.
  • Transfer cells + DNA onto agar.
  • Add 8-10 glass beads, shake, then discard beads.
  • Incubate plates at 37°C overnight and retrieve next day.