Chemicon 试剂盒的说明

TROUBLESHOOTING

No products are visible in any lane.

1. Potential Problem: PCR amplification is not initiated.
Recommendations:
a. Confirm that all PCR components were added to the reaction tube.
b. If performing hot start PCR using AmpliTaqGold®, verify the initial
denaturation/activation time of 12 minutes at 95°C.
c. For all other hot start methods, confirm the proper use of the reagents.

2. Potential Problem: Experimental DNA samples were degraded prior to chemical modification.
Recommendation: Purify the genomic DNA again and repeat the chemical modification. Methylated and Unmethylated Control DNAs (unmodified) are available from Chemicon for use as a positive controls.

3. Potential Problem: DNA samples were degraded after modification.
Recommendation: If chemically modified experimental DNA samples were stored at �20°C for more than two months prior to PCR, repeat the chemical modification on new genomic DNA samples. Do not store DNA in a selfdefrosting freezer.

The only lanes containing a PCR product are from those DNA samples amplified with the W primer set.

1. Potential Problem: Chemical modification of the experimental DNA samples did not work.
Recommendations:
a. DNA Modification Reagent and 3 M NaOH stock must be freshly prepared prior to each use.
b. Sodium bisulfite modifies only single-stranded DNA. Double stranded DNA should be denatured in 200 mM NaOH prior to modification. Some highly G/C rich sequences may denature better if a restriction digestion step is inserted prior to denaturation. The cuts should be carefully targeted outside the intended template sequence.

2. Potential Problem: DNA was degraded during modification.
Recommendation: Incubation of DNA at pH 5 unavoidably causes some damage. Incubation in DNA Modification Reagent at 50°C for over 16 hours could cause some methylated cytosines to be converted to thymidines.

3. Potential Problem: Some DNA sequences form secondary structures that are resistant to chemical modification.
Some DNA samples are partially or randomly methylated, and after modification, these might not bind the MSP primers under the recommended PCR conditions.
Recommendation: These possibilities can be explored by cloning, amplifying and sequencing the modified DNA. The annealing temperature of the MSP reaction may be lowered by 3-6°C to increase primer binding, although this may promote artifacts due to mispriming.

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W primer set produces an amplification product in some or all experimental samples, in addition to an amplification product from the U or M primer set.
1. Potential Problem: Chemical modification of the experimental DNA sample is incomplete.
Recommendation: This will not jeopardize the validity of the assay as long as a product is also produced using the U or M primer set.
U and M primer sets are both producing bands in some DNA samples.
Potential Problem: Sample heterogeneity.
Recommendations: The DNA sample may have been derived from heterogeneous original cell types, containing both methylated and unmethylated DNA. This is common with many tumor samples.
U or M primer sets are producing bands in all samples,including the "no DNA" controls.
Potential Problem : PCR reagents are contaminated with amplification products.
Recommendations: see Sec.V. Appendix: Laboratory Setup and Precautions
a. Use fresh aliquots of every PCR component (i.e. dNTPs, buffer, etc).
b. Use separate sets of pipettes for each liquid dispensing.
c. Devote a work area to pre- and post-amplification procedures.
d. Always use aerosol-resistant pipette tips.
e. Always use a clean labcoat and gloves.
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V. APPENDIX
Considerations for MSP
Primer Design

Methylation Specific PCR consists of chemical modification followed by amplification. Chemical modification creates the sequence differences between the methylated and unmethylated DNA. Ideally, three sets of primers should be designed to anneal to the DNA, based upon these sequence differences. One primer set (U) will anneal to unmethylated DNA that has undergone a chemical modification. A second primer set (M) will anneal to methylated DNA that has undergone a chemical modification. A third primer set (W) will anneal to any DNA (unmethylated or methylated) that has NOT undergone chemical modification, hence, the “wild type”, or W. This serves as a control for the efficiency of chemical modification.
The standard rules for primer design apply toward the creation of MSP primers.
The primers should be approximately 20-21 bp in length and should possess similar dissociation temperatures. The product produced with each set of primers should be 100-200 bp in size. Internal secondary structure should be avoided. In order to minimize primer dimer formation, primers should not be complementary, especially at the 3’ end. Since most CpG islands are located within the gene promoter, the optimal area for primer selection is often the most G-C rich region closest to the transcription start site. Discrimination between methylated and unmethylated sequences by MSP seems to be greatest when the 3’ ends of the primers are most different from one another. Alternatively, Chemicon has introduced CpG Ware™ Primer Design Software, a functional program that selects oligonucleotide primers from a sequence file for methylation-specific PCR (MSP) analysis. The interactive program will select and generate the most suitable primer sets that may potentially be used for MSP analysis to discriminate between methylated and unmethylated DNA sequences
of interest. The user is presented with the native and modified DNA sequences, suggested primer sets (1 to 4) and the corresponding amplicon sizes and nudelotide positions. Please visit the Chemicon website at www.chemicon.com for access to of the CpG Ware™ software.