Assignment for laboratory 10
- 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)
- 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
- 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
- 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 dNTP | Tail Length | |||
| dA | dC | dG | dT | |
| 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.