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- Slot Blot
Dilute aliquot of each sample in 6xSSC. Denature at 100°C ~10 minutes. Place immediately on ice. Apply samples to Nytran. Wash 2x with 6xSSC. UV crosslink filter prior to hybridization.
- Southern Blot
Usually need to digest at least 1/2 of Pellet samples to see a signal; therefore, aliquot 75 µL to new Eppendorf tube and reduce volume in speed vac. Alternatively, if Southern blot analysis is anticipated, resuspend Pellets in a smaller volume initially. For Totals and Sups, digest an amount of chromatin solution equivalent to 1/5 to 1/10 amount used for Pellets.
For CEN3, AluI is the diagnostic digest. Digest for several hours to overnight. Add RNase to bluejuice for Totals and Sups prior to loading onto gel. Run a 2.5% agarose gel. Transfer to GeneScreen. (I leave out the HCl treatment when preparing gels for transfer.). UV crosslink prior to hybridization.
- PCR
I use 3 µL of each sample to program a 50 µL PCR reaction. (Note: Given the volumes used to resuspend various samples, the chromatin solution equivalent for Total is 1/10 that used for Pellet). Controls include no DNA and plasmid or good genomic DNA as a positive control.
| Reaction Conditions (in 50 µL): |
Program: |
| DNA (3 µL) |
95°C, 3 minutes |
| 1x Taq Bfr. |
|
| 1.5 mM MgCl2 |
95°C, 30 seconds |
| 1 µM each primer |
Tm-5°C, 45 seconds |
| 0.2 mM dNTPs |
72°C, 60 seconds |
| 0.5 µL Taq |
|
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Amplify for 24 cycles, total. |
|
72°C, 5 minutes |
|
4°C | If the experiment requires much PCR analysis, pellets can be diluted 1:4 in TE, with an equivalent dilution of totals (1:36). 3 µl of diluted template with 26 cycles of PCR will remain within a linear range for quantitation.
Add 5x Bluejuice. Analyze 1/3 to 1/2 reaction on 2.5% agarose gel or 8% PAGE. Remember to be quantitative!!
- NuSieve GTG Agarose Gels
PCR products are run on a 2.5% NuSieve GTG agarose gel (this works well for the typical size range of ChIP PCR products- 150-500 bp. For the large gel trays (20cm x 25cm), 200ml of 2.5% agarose is required. Weigh out agarose and place in a large flask to prevent boiling over during microwaving, and add 1X TBE buffer. The manufacturer recommends using cold buffer, but I have not found this to be critical. Stir the agarose solution for 20 minutes before microwaving. Weigh the flask before microwaving so that water lost during microwaving can be added back to the agarose before pouring. Use the following microwave settings: 3 minutes at power level 5, 30-45 at seconds highest power level. Stir the agarose to cool before pouring. Reweigh the flask and add water lost during heating. Just before pouring (I generally pour the gels in the cold), add ethidium bromide to a final concentration of 0.15 µg/ml (3µl of 10 mg/ml stock).
After 30-45 minutes in the cold, place gel in the gel box and add 2 L of 1X TBE containing 0.15 µg/ml ethidium bromide. Let the gel sit for a while before removing combs. I typically run my gels at 120V for 90 minutes. Using these conditions, the current during the run generally begins at 70 mA and finishes at 80 to 90 mA. Avoid running gels at voltages that generate currents greater than 90 mA to prevent diffuse bands and heating of the agarose.
Reagents Needed for ChIP
| 37% formaldehyde stock solution |
1 M DTT stock |
| 1 M TRIS, pH 9.4 stock |
1 mg/mL Oxalyticase stock or 10 mg/mL Zymolyase stock |
| PMSF, pepstatin A (1 mg/ml in 100% MeOH), leupeptin (1mg/ml in sterile water) stocks |
|
HEPES/sorb
20 mM HEPES, pH 7.4
1.2 M sorbitol
|
250 mL:
5 mL 1 M HEPES
150 mL 2 M sorbitol |
PIPES/sorb
20 mM PIPES, pH 6.8
1 mM MgCl2
1.2 M sorbitol |
250 mL:
5 mL 1 M PIPES
0.25 mL 1 M MgCl2
150 mL 2 M sorbitol |
| PBS |
|
Triton/HEPES Wash
0.25% Triton X-100
10 mM EDTA
0.5 mM EGTA
10 mM HEPES, pH 6.5 |
250 mL:
3.13 mL 20% Triton X-100
5 mL 0.5 M EDTA
0.5 mL 0.25 M EGTA
2.5 mL 1 M HEPES |
NaCl/HEPES Wash
200 mM NaCl
1 mM EDTA
0.5 mM EGTA
10 mM HEPES, pH 6.5 |
250 mL:
10 mL 5 M NaCl
0.5 mL 0.5 M EDTA
0.5 mL 0.25 M EGTA
2.5 mL 1 M HEPES
|
SDS Lysis Buffer
1% SDS
10 mM EDTA
50 mM TRIS, pH 8.1 |
100 mL:
10 mL 10% SDS (UltraPure)
2 mL 0.5 M EDTA
5 mL 1 M TRIS
|
IP Dilution Buffer (for 1:9 dilution)
0.01% SDS
1.1% Triton X 100
1.2 mM EDTA
16.7 mM TRIS, pH 8.1
167 mM NaCL |
250 mL:
0.25 mL 10% SDS
13.8 mL 20% Triton X 100
0.6 mL 0.5 M EDTA
4.2 mL 1 M TRIS, 8.1
8.35 mL 5 M NaCl
|
Sonicated lambda DNA. Store at 4°C with 0.1% azide.
|
|
| Protein A Sepharose Bead Buffer: |
25 mL:
25 mL TE
25 mg BSA (Fraction V, powder)
25ul 10 % azide
|
TSE-150 Wash (= IP conditions)
0.1% SDS
1% Triton X-100
2 mM EDTA
20 mM TRIS-HCl, pH 8.1
150 mM NaCl |
250 mL:
2.5 mL 10% SDS
12.5 mL 20% Triton
1.0 mL 0.5 M EDTA
5.0 mL 1 M TRIS, 8.1
7.5 mL 5 M NaCl
|
TSE-500 Wash (Optional)
0.1% SDS
1% Triton X-100
2 mM EDTA
20 mM TRIS-HCl, pH 8.1
500 mM NaCl |
250 mL:
2.5 mL 10% SDS
12.5 mL 20% Triton
1.0 mL 0.5 M EDTA
5.0 mL 1 M TRIS, 8.1
25 mL 5 M NaCl
|
LiCl/Detergent Wash
0.25 M LiCl
1% NP-40
1% DOC
1 mM EDTA
10 mM TRIS-HCl, pH 8.1 |
250 mL:
2.65 g LiCl (42.39)
25 mL 10% NP-40
50 mL 5% DOC
0.5 mL 0.5 M EDTA
2.5 mL 1 M TRIS, 8.1
|
TE, pH 8.0
|
|
Bead Elution Buffer
1% SDS/0.1 M NaHCO3 |
50 mL:
5 mL 10% SDS
5 mL 1M NaHCO3
|
5x Proteinase K Buffer
50 mM TRIS, pH
25 mM EDTA
1.25 % SDS |
10 mL:
0.5 mL 1 M TRIS
0.5 mL 0.5 M EDTA
1.25 mL 10 % SDS
|
Proteinase K Solution (Boehringer Mannheim, 1413 783), ~18.6 mg/mL
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Glycogen stock
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