| Contributor: |
The Laboratory of Jim Kadonaga at the University of California, San Diego
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| Topoisomerase I is a chromatin-associated enzyme that alters the topology of DNA. This assay measures the ability of the purified enzyme to relax supercoiled plasmid DNA based on the linking number of the circular DNA. |
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1. Combine the following
5 μl of 10X Topo I Reaction Buffer (see Hint #1)
5 μl of 0.1 μg/ul supercoiled pUC DNA (or equivalent plasmid) in TE buffer
39 to 35 μl of ddH2O
1 to 5 μl of purified Topoisomerase I sample (from P11 phosphocellulose column fractions. See Protocol on Preparation of Topoisomerase I from Drosophila Embryos; see Hint #2)
Total reaction volume is 50 μl
Set up a positive control with a known Topoisomerase I standard and a negative control containing buffer in place of the Topoisomerase I sample.
2. Incubate at 30°C for 30 min.
3. Stop the reaction by adding 15 μl of Stop Solution.
4. Pour a 0.8% Agarose Gel and pour a 0.8% Agarose Gel Chloroquine (see Protocol on Agarose Gel Electrophoresis of DNA and see Hint #3).
5. Add 4.6 μM Chloroquine to the running buffer of the 0.8% Agarose Gel with Chloroquine.
6. Load 10 μl of the samples onto both gels. Be sure to also run supercoiled DNA and relaxed DNA as negative and positive controls, respectively.
7. Electrophorese the samples at 100 V.
8. Stain the gels in Ethidium Bromide Solution. Destain the gels in ddH2O.
9. Visualize the gels with a UV transilluminator and analyze.
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| Ethidium Bromide Solution |
| 0.5 μg/ml Ethidium Bromide (CAUTION! see Hint #4)
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| 0.8% Agarose Gel With Chloroquine |
| When the Agarose has cooled to 55°C, add 8 μl of Chloroquine Stock (final concentration 4.6 μM)
Make up in 50 ml of 1X TAE
Microwave the Agarose in 1X TAE to dissolve the Agarose granules
0.8% (w/v) Agarose
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| TAE Buffer (50X) |
| 57.1 ml/liter Glacial Acetic Acid
Adjust to pH 8.5
242 g/liter Tris base
37.2 g/liter EDTA, disodium salt
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| Chloroquine Stock |
| 15 mg/ml Chloroquine Diphosphate (Sigma) (CAUTION! see Hint #4)
Store at 4°C in the dark (the solution is stable for months)
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| Stop Solution |
| 5% (w/v) SDS
25% (v/v) Glycerol
It may be necessary to warm up the solution just before use in order to dissolve the SDS
0.25 μg/ml Bromophenol Blue
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| TE Buffer |
| 10 mM Tris-Cl, pH 7.5
1 mM EDTA
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| Topo I Reaction Buffer (10X) |
| 5 mM DTT
0.5 mg/ml Acetylated BSA
500 mM Tris-Cl, pH 7.5
Store at -20°C
100 mM MgCl2
2 mM EDTA
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DTT
Magnesium Chloride
Glycerol
Bromophenol Blue
SDS
Agarose
DNA, Supercoiled pUC
EDTA
Glacial Acetic Acid
Acetylated BSA
Chloroquine
Tris
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1. Because the Topoisomerase I P11 phosphocellulose column fractions will have a salt concentration of 1.0 to 1.5 M NaCl, the addition of the purified protein will significantly increase the salt concentration in the reaction. Therefore, the assay buffer has been adjusted accordingly.
2. Typically about 1 to 5 μl of the P11 phosphocellulose column fractions will be sufficient for the assay. One μl of the topoisomerase I sample will result in a final NaCl concentration of 20 mM to 30 mM in the reaction.
3. Chloroquine is a DNA intercalating agent that introduces positive supercoiling into the DNA and thus allows better resolution by electrophoresis of highly negatively supercoiled DNA.
4. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
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