Haynes Lab:Notebook/Engineering PC-TFs/2013/03/27

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3/27/2013

  • 1. Kozak,V0120 + BL01
  • 2. Kozak,V0120 + BL05
  • 3. Kozak,V0120 + BL09
  • 4. Kozak,V0120 + BL12
# Biobrick ng/µL
1 hPCD : mCh : SP1AB (BL01) 18.834
2 fshPCD : mCh : SP1AB (BL05) 14.471
3 flyPCD : mCh : SP1A (BL09) 7.302
4 hPCD : BL01 (BL12) 10.84








µL of insert = (length insert/lengthvector *2*25 ng vector/concentration insert)
1 2 3 4 5
DNA Insert1.41.82.02.5--
DNA Vector (Kozak)0.60.60.60.60.6
T4 Ligase11111
Lign Buffer (2x)55555
dH2O2.01.61.4.93.7
10 µL10 µL10 µL10 µL10 µL
  • In a .5 mL tube, pipette the ligation buffer first.
  • Then water, DNA insert, vector, and T4 ligase.
  • Once completed, allow them to incubate for 10 minutes.
  • Meanwhile, get ice bucket and allow competent E. coli cells to thaw for five minutes.
  • Then follow transformation process:

Transformation Process

  • Warm five 100 μg/mL Amp agar plates at 37 °C
  • Thaw fresh tube of DH5α Turbo cells on ice
  • Add 30 μL of DH5α Turbo cells to DNA + dH2O
  • Include #5 tube, water only, no DNA (negative control)
  • Incubate cells + DNA on ice for 10 min.
  • Label pre-warmed plates
  • Transfer cells + DNA onto agar
  • Add 10 - 15 sterile glass beads, shake, discard beads
  • Incubate plates at 37 °C overnight
Ligation attempt 3/27/2013
Ligation attempt 3/27/2013
  • No colonies on any of the plates. Try again.



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