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The Laboratory of Donald Rio at the University of California, Berkeley
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1. Combine the following reaction ingredients:
(X) μl transposase-containing fraction
(Y) μl P element-containing plasmid DNA (approximately 100 ng)
(Z) μl HGKED/BSA
The final volume should be 6 μl.
The amounts of the transposase-containing fraction and the P element need to be determined empirically.
You will want to make sure that the final KCl concentration is below 35 mM (see Hint #2).
2. Incubate for 15 min on ice to pre-bind.
3. Initiate the reaction by adding the following components as a cocktail:
4.9 μl of 1X HGED (0.35X final concentration)
0.2 μl of 10 mM MgCl2 (1 M final concentration)
0.4 μl of 2 mM GTP (100 mM final concentration )
8.5 μl of ddH2O
The final volume of 14 μl is added to the 6 μl pre-hybridization volume for a final reaction volume of 20 μl.
4. Incubate at 27°C for 2 hr.
5. Stop the reaction by adding 125 μl of Footprint Stop Solution and 1.5 μl of 10 mg/ml Proteinase K.
6. Incubate at 37°C for 30 min.
7. Add 125 μl of Extraction Buffer, mix well by inversion, microcentrifuge to separate phases and save the aqueous phase (upper layer).
8. To the aqueous phase, add 450 μl of 100% Ethanol, mix well by inversion and microcentrifuge to pellet the DNA bound protein.
9. Decant the supernatant and to the pellet add 450 μl of 70% Ethanol, mix well by inversion and microcentrifuge to pellet the DNA bound protein.
10. Decant the supernatant, dry the pellet by inverting the tubes over a paper towel and allow the sample to air dry for 2 to 3 min.
11. Resuspend in 10 μl of TE/RNase.
12. Incubate for 15 min at room temperature to digest the RNA.
13. Load 5 μl onto a 1% agarose 1XTBE gel (see Agarose Gel Electrophoresis Protocol) and electrophorese until the bromophenol blue dye has migrated about two-thirds down the gel.
14. Blot the DNA from the gel onto Hybond N membrane (see Southern Blotting Protocol using Hybond N protocol).
15. Stratalink the blot, prehybridize and use a random hexamer-labeled P element DNA fragment as a probe. Hybridize as usual at 65°C (see Hybridization Protocols) and expose to film at -80°C (see Protocol on Autoradiography).
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| TE/RNase |
| Prepared in TE buffer
100 μg/ml RNase A
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| TE Buffer |
| 10 mM Tris
pH 8.0
1 mM EDTA
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| 70% (v/v) Ethanol |
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| Extraction Buffer |
| 1 part Isoamyl Alcohol (CAUTION! see Hint #1)
24 parts Chloroform (CAUTION! see Hint #1)
25 parts Phenol (CAUTION! see Hint #1)
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| Proteinase K |
| Prepared in ddH2O
10 mg/ml Proteinase K
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| Footprint Stop solution |
| 0.2 M NaCl
1% (w/v) SDS
20 mM EDTA
Prepared in ddH2O.
250 μg/ml Yeast RNA
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| HGKED (1X) |
| (X)* KCl
Add to final concentration of 0.5 mM DTT and 0.2 mM PMSF just before use.
100 μg/ml Bovine Serum Albumin (Pentex)
1 mM EGTA
1 mM EDTA
25 mM HEPES-KOH, pH 7.6
20% (v/v) Glycerol
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| HGED (1X) |
| 1 mM EGTA
1 mM EDTA
20% (v/v) Glycerol
25 mM HEPES-KOH, pH 7.6
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| HGKED/BSA |
| Add to final concentration of 0.5 mM DTT and 0.2 mM PMSF (CAUTION! see Hint #1) just before use.
(X)* KCl
100 μg/ml Bovine Serum Albumin (Fraction V)
*(X) the concentration of KCl will vary depending on the salt concentration of the transposase-containing fraction. The salt concentration MUST BE BELOW 35 mM (see Hint #2).
1 mM EGTA
1 mM EDTA
20% (v/v) Glycerol
25 mM HEPES-KOH, pH 7.6
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SDS
HEPES
EDTA
Potassium Chloride
Sodium Chloride
Oligonucleotide
RNase A
Ethanol
EGTA
Bovine Serum Albumin
Glycerol
Isoamyl Alcohol
Yeast RNA
Chloroform
Phenol
Proteinase K
Tris
PMSF
DTT
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1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. The potassium (K) salt concentration must be kept below 35 mM for the pre-binding step and the cleavage step.
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