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

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*5. hPCD (3386) only
*5. hPCD (3386) only


Assembly Scheme
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Revision as of 19:35, 17 February 2012

Engineering PC-TFs <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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Ligation/Assembly

  • Weight 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 (3386) + KAH204 (2328)
  • 2. hPCD (3386) + KAH205 (1251)
  • 3. hPCD (3386) + KAH206 (1551)
  • 4. hPCD (3386) + KAH225 (1251)
  • 5. hPCD (3386) only
' 1 2 3 4 5
DNA Insert 1.3 0.4 0.5 0.3 -----------
DNA Vector 0.3 0.3 0.3 0.3 0.3
T4 Ligase 1 1 1 1 1
Lign Buffer (2x) 5 5 5 5 5
dH2O 2.4 3.3 3.5 3.7 4
30 µL 30 µL 30 µL 30 µL 30 µL


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