User:Daniel A. Vargas/Notebook/General lab notebook/2015/08/04

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ASSEMBLY of MV10, ATF2, and Gal4DB/mCherry

  • LCR Assembly


LCR Assembly


PNK treatment Overview: Mix double-stranded DNA fragments, treat with PNK to add phosphate groups

Procedure:

  • Dilute the purified dsDNA to 30 fmol/μL (30 nM)
    • The volume of purified DNA (x) you will need to dilute in a final volume of 50 μL = length in bp ÷ measured ng/μL * 30 fmol/μL * 650 fg/fmol dsDNA ÷ 1,000,000 fg/ng * 50 μL final volume.
    • Formula: x μL = length in bp ÷ measured ng/μL * 0.0195 ng/μL * 50μL
  • Use 2.0 μL of each diluted dsDNA per 10 μL PNK reaction
  • Mix the appropriate amount of dsDNA fragments together and treat with Polynucleotide kinase to add 5'-phosphates (see table below)


PNK reaction

Reagent Volume
30 fmol/μL MV10 2.0
30 fmol/μL Gal4DB-mCh 2.0
30 fmol/μL ATF2 2.0
10x T4 Ligation buf (NEB) 1.0
T4 PNK (NEB) 0.5
dH2O 2.5 μL
  10.0 μL
  • Incubate at 37°C/ 30 min.
  • Heat-inactivate PNK at 65°C/ 20 min.




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