Haynes Lab:Notebook/Engineering PC-TFs/2012/02/16

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Ligation/Assembly

  • Weigh gel slices.
  • Add three times amount of volume to each tube of gel (600 µL)
  • Incubate at 55°C for 5-10 minutes.
  • Centrifuge for a minute at 17,000 x g.
  • Add 20 µL of wash buffer.
  • Don't dump supernatent and centrifuge for one minute again.
  • Place spin columns in 1.5 mL tubes.

Measure concentration of each fragment:

  • Load Gen 5 2.0 software. Go to the Task Manager-Take 3 Application
  • Go to Nucleic Acid Quantification.
  • Pipette 2 µL of each DNA sequence into Take 3 plate.
  • Place plate in EPOCH plate reader.

Results:

Molarity of DNA Fragments
1. KAH01 96.477 ng/µL
2. KAH03 122.149 ng/µL
3. KAH04 80.182 ng/µL
4. KAH204 27.054 ng/µL
5. KAH205 47.105 ng/µL
6. KAH206 45.217 ng/µL
7. KAH225 66.047 ng/µL


  • For all ligations, 25 ng of DNA vector = .3 µL
  • There is a 2X molar ratio of insert:vector
µL of insert = (length insert/lengthvector *2*25 ng vector/concentration insert)

Assembly Scheme:

Number beside vector and insert is number of base pairs

  • 1. hPCD (186) + KAH204 (2328)
  • 2. hPCD (186) + KAH205 (1251)
  • 3. hPCD (186) + KAH206 (1551)
  • 4. hPCD (186) + KAH225 (1251)
  • 5. hPCD (186) only
' 1 2 3 4 5
DNA Insert1.30.40.50.3-----------
DNA Vector0.30.30.30.30.3
T4 Ligase11111
Lign Buffer (2x)55555
dH2O2.43.33.53.74
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 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.
  • Incubate cells + DNA on ice for 5 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



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