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Could your labeled dCTP be used for DNA sequencing? Describe a method of how it could be used, or describe several DNA sequencing methods which where it would be ineffective and explain why (look at slide 56, 6200J20.pdf)

Assignment for laboratory 10

  1. Could your labeled dCTP be used for DNA sequencing? Describe a method of how it could be used, or describe several DNA sequencing methods which where it would be ineffective and explain why (look at slide 56, 6200J20.pdf)
    1. Hint: Look at the DNA sequencing methods used in this class. Which would be most suitable for time-resolved fluorescence labels given their relatively slow imaging times?
  • Did the absorbance spectrum indicate that the dCTP was labeled? Can you estimate the fraction of dCTP that was labeled? (look at IEX_Chromatography_Lab results file)
    • *To determine approximate amount of DNA (ng/pmol), multiply the number of base pairs by 0.66. Example: 300 bp x 0.66 = 198 ng/pmol. For 5.0 pmols multiply by 5, resulting in 990 ng/5 pmol.
    • Hint: to estimate the fraction, look at the absorbance spectra of the peak fractions and the relative areas under the peaks of the ion exchange chromatograph that were given to you
  1. What is the concentration of labeled dCTP? How was it determined? What is the protocol for the tailing reaction? (look at TailingReaction.pdf file)
  • Describe a FRET assay using the labeled dCTP as a donor or acceptor.
  • Compare the tailing reaction with several other common methods for making a probe that could incorporate your labeled dCTP ( look at slide 33, 6200J20.pdf).

Protocol

  1. Mix: 
    a. 5.0 μl (10X) TdT Buffer
    b. 5.0 μl (2.5 mM) CoCl2 solution provided
    c. 0.2 μl of 5.0 pmols DNA 47 bases single standed
    d. 39.3 μl 8.7 μM dCTP-DTPA
    e. 0.5 μl Terminal Transferase (20 units/μl)

*To determine approximate amount of DNA (ng/pmol), multiply the number of base pairs by 0.66. Example: 300 bp x 0.66 = 198 ng/pmol. For 5.0 pmols multiply by 5, resulting in 990 ng/5 pmol.

The table below can be used as a guide (values are approximate and are given for a 30 minutes incubation at 37°C in the recommended buffer).

The rate of addition of dNTP’s and thus the length of the tail is a function of the ratio of 3´ DNA ends: dNTP concentration, and also which dNTP is used.

DNA Tailing Guide:

pmols 3′ ends pmols dNTPTail Length 
 dAdCdGdT
1:100 1-5 1-3 1-3 1-5
1:1,000 10-20 10-20 5-10 10-20
1:5,000 100-300 50-200 10-25 200-300
  • Incubate at 37°C for 30 minutes.
  • Stop the reaction by heating to 70°C for 10 minutes, freezing, or by adding 10 µl of 0.2 M EDTA (pH 8.0).

4. Determine concentration of DTPA-dCTP with molar extinction coefficients of 22000 1/(M cm) @ 243 nm or 9000 1/(M cm) @ 293 nm.

5. The ssDNA oligonucleotide, C682-1 has a molar extinction coefficients of 469270 1/(M cm) @ 260 nm and is 47 nucleotides.

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