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The following Cold Spring Harbor Laboratory Press protocols are brought to you by BioSupplyNet in a partnership with Sigma-Aldrich. A list of products is available to use in each protocol.
View the protocol on BioSupplyNet.com. |
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Selected Protocol from "Proteins and Proteomics: A Laboratory Manual,", David R. Goodlett, Eugene C. Yi, and Philippe Mottay.
View the protocol
"PCR-Based Analysis of Immunoprecipitated Chromatin" at BioSupplynet.com
Alexandre Wagschal, Katia Delaval, Maëlle Pannetier, Philippe Arnaud, and Robert Feil - Institute of Molecular Genetics, CNRS and University of Montpellier, 34293 Montpellier, France
ABSTRACT
After chromatin immunoprecipitation (ChIP), different PCR-based approaches can be used to determine how much DNA is precipitated at a locus of interest. Real-time PCR amplification is often the preferred technique. One can also use duplex PCR amplification, which is the coamplification of a fragment from the region of interest and a control fragment (e.g., the actin gene, or the tubulin gene). This approach allows for estimating relative levels of specific histone modifications along chromosomal domains. For allele-specific studies (for instance, on dosage-compensation mechanisms or on genomic imprinting), electrophoretic detection of single-strand conformation polymorphisms (SSCP) or similar strategies such as hot-stop PCR can differentiate PCR products that represent the silent allele from those amplified from the active allele. If a polymorphic restriction site is present in one allele and absent in the other, the method of choice is hot-stop PCR. If no polymorphic restriction sites are available, but there are single nucleotide polymorphisms (SNPs) that distinguish the alleles of interest, the best approach is to separate the PCR products derived from the two different alleles using SSCP. In SSCP, it is possible to discriminate denatured PCR products derived from one allele or the other because the secondary structure of each single strand will be directly dependent on the sequence itself. Hence, in nondenaturing gel conditions, each single strand will migrate differently. These four PCR-based methodologies to analyze immunoprecipitated chromatin (real-time PCR, duplex PCR, hot-stop PCR, and SSCP) are presented here.
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Products Available for this Protocol
| Protocol Material Description |
Product # |
Product Name |
Add to Cart |
| 100-bp DNA step ladder |
D5042 |
123 bp Ladder, buffered aqueous solution |
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| Acrylamide solution for SSCP gels (2X) |
01697 |
Acrylamide solution, BioChemika, for molecular biology, 40% in H2O |
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| Acrylamide/bisacrylamide (29:1 ratio; 40% stock solution) |
A2792 |
Acrylamide/Bis-acrylamide, 29:1 (ratio), DNase, RNase and protease, none detected, Molecular Biology |
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| Agarose |
A9539 |
Agarose, for routine use |
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| Ammonium persulfate (APS; 10%, w/v) |
A9164 |
Ammonium persulfate, for molecular biology, powder, ≥98% |
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| dNTPs |
DNTP100 |
Deoxynucleotide Set, 100 mM |
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| Xylene cyanol FF |
X4126 |
Xylene Cyanol FF for molecular biology |
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| Bromophenol blue |
B5525 |
Bromophenol Blue sodium salt for electrophoresis |
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| Ethidium bromide solution |
E1385 |
Ethidium bromide solution, for molecular biology, 500 µg/mL in H2O, suitable for gel electrophoresis and DNA isolation procedures |
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| PCR amplification buffer (with Taq) |
View the entire line of PCR amplification mixes |
| Restriction endonuclease |
View the entire line of restriction endonucleases |
| Restriction endonuclease buffer (10X) (supplied with the restriction endonuclease) |
View the entire line of restriction endonuclease buffer |
| Formamide |
F7508 |
Formamide, for molecular biology, ≥99.5% (GC), liquid |
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| NaOH |
72068 |
Sodium hydroxide solution, BioChemika Ultra, for molecular biology, 10 M in H2O |
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| Taq DNA polymerase (5 U/µl) |
D1806 |
Taq DNA Polymerase from Thermus aquaticus, with 10× reaction buffer containing MgCl2, recombinant, expressed in Escherichia coli |
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| TBE buffer |
T4073 |
Tris-Borate-EDTA buffer, working solution, Biotechnology Performance Certified |
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| Template DNA |
D4642 |
Deoxyribonucleic acid from human placenta, Genomic |
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Product Association Disclaimer: The Sigma-Aldrich products listed for this specific protocol were selected either to match or to supplement the products listed within the actual protocol. The products/reagents from Sigma-Aldrich have been qualified for usage, but may not have been validated for this specific application. Please refer to the detailed product description on the usage of specific products of interest.
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