FIGURE 1. Amplification plot (left) and regression curve (right) of a serial dilution of input DNA samples. Input DNA was prepared and purified from HCT 116 cells using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. Quantitative PCR was performed with 20 (red line), 4 (orange line), 0.8 (purple line), and 0.016 ng (blue line) of input DNA and SimpleChIP® Human RPL30 Exon 3 Primers #7014. Data is shown for two replicates.Learn more about how we get our images
FIGURE 2. Corresponding single melt peak of primers. Quantitative PCR was performed with input DNA sample from HCT 116 cells prepared by SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005 and either SimpleChIP® Human α Satellite Repeat Primers #4486 (red line), SimpleChIP® Human RPL30 Exon 3 Primers #7014 (orange line), SimpleChIP® Human CaMK2D Intron 3 Primers #5111 (purple line), or human c-MYC promoter primers (blue line). Data for two replicates is shown for each primer set.Learn more about how we get our images
FIGURE 3. Quantitation of ChIP DNA enriched using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HCT 116 cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 (left panel), TCF4/TCF7L2 (C48H11) Rabbit mAb #2569 (right panel), and Normal Rabbit IgG #2729 (both panels). The enriched DNA was quantified by real-time PCR using either SimpleChIP® Human RPL30 Exon 3 Primers #7014, human c-Myc promoter primers, SimpleChIP® Human CaMK2D Intron 3 Primers #5111, or SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.Learn more about how we get our images
|Primer length:||24 nucleotides|
|Amplicon size:||80 to 160 bp|
|Reagent||Volume for 1 PCR Reaction (18 μl)|
|Nuclease-free water||6 μl|
|Primers (5 μM)||2 μl|
|SimpleChIP® Universal qPCR Master Mix (2X) #88989||10 μl|
|a.||Initial Denaturation||95°C 3 min|
|b.||Denature||95°C 15 sec|
|c.||Anneal and Extension||60°C 60 sec|
|d.||Repeat steps b and c for a total of 40 cycles.|
|e.||Melt Curve step from 60-95°C.|
Quantify experimental samples relative to an input control or standard curve using the software provided with the real-time PCR machine. Alternatively, one can calculate the IP efficiency manually using the Percent Input Method and the equation shown below. With this method, signals obtained from each immunoprecipitation are expressed as a percent of the total input chromatin.
Percent Input = 2% x 2(C[T] 2%Input Sample – C[T] IP Sample)
C[T] = CT = Average threshold cycle of PCR reaction
|1. qPCR traces show low or no amplification.||Poor ChIP-DNA sample or primer design.||ChIP DNA should be purified using a DNA purification column or equivalent method. Unpurified DNA gives very poor amplification.|
|Low or no amplification of purified input DNA is indicative of a poor primer set and optimization or redesign of the primer set may be needed.|
|Efficient amplification of input DNA and absence of amplification of ChIP sample DNA is indicative of a failed ChIP immunoenrichment.|
|Reagent added improperly or omitted from qPCR assay.||Verify all steps of the protocol were followed correctly.|
|Incorrect cycling protocol.||Refer to the proper qPCR cycling profile in this protocol.|
|Incorrect reporter dye selected for the qPCR thermal cycler.||Select FAM/SYBR® channel on the qPCR instrument.|
|Reagents are contaminated or degraded.||Confirm the expiration dates of the kit reagents and verify proper storage conditions. Rerun the qPCR assay with fresh reagents.|
|2. Inconsistent qPCR traces for triplicate data.||Improper pipetting during qPCR assay set-up.||Ensure proper pipetting techniques.|
|qPCR plate film has lost its seal, causing evaporation in the well. The resulting qPCR trace may show significantly different fluorescence values relative to its replicates.||Ensure the qPCR plate is properly sealed before inserting into the qPCR thermal cycler. Exclude problematic trace(s) from data analysis.|
|Poor mixing of reagents during qPCR set-up.||Make sure all reagents are properly mixed after thawing them.|
|Bubbles cause an abnormal qPCR trace.||Avoid bubbles in the qPCR plate. Centrifuge the qPCR plate prior to running it in the thermal cycler. Exclude problematic trace(s) from data analysis.|
|3. DNA standard curve has a poor correlation coefficient/ efficiency of the DNA standard curve falls outside the 90–110% range||Presence of outlying qPCR traces.||Omit data produced by qPCR traces that are clearly outliers caused by bubbles, plate sealing issues, or other experimental problems.|
|Improper pipetting during qPCR assay set-up.||Ensure that proper pipetting techniques are used.|
|Reaction conditions are incorrect.||Verify that all steps of the protocol were followed correctly.|
|Bubbles cause an abnormal qPCR trace.||Avoid bubbles in the qPCR plate. Centrifuge the qPCR plate prior to running it in the thermal cycler.|
|Poor mixing of reagents.||After thawing, make sure all reagents are properly mixed.|
|Threshold is improperly set for the qPCR traces.||Ensure the threshold is set in the exponential region of qPCR traces. Refer to the real-time instrument user manual to manually set an appropriate threshold.|
|4. Melt curve shows multiple peaks or different peaks for low input samples.||Non-template amplification is occurring.||Compare melt curve of NTC to samples. Similar melt curves for DNA samples and NTC are indicative of non-template amplification.|
|Non-specific amplification is occurring, or infrequently, denaturation of a single species can occur in a biphasic manner, resulting in two peaks.||Redesign primers with a Tm of 60°C or determine the optimal annealing temperature of the primers.|
|Perform a primer matrix analysis to determine optimal primer concentrations.|
|5. No template control qPCR trace shows amplification, NTC CT is close to or overlapping lowest input DNA titration standards.||Reagents are contaminated with DNA (Melt curve of NTC matches melt curve of Input DNA standards).||Replace all stocks and reagents. Clean equipment and setup area with a 10% chlorine bleach.|
|Primers produce nonspecific amplification (Melt curve of NTC does not match melt curve of Input DNA standards).||Redesign primers with a Tm of 60°C or use qPCR primer design software.|
posted November 2017
Protocol Id: 1584
The SimpleChIP® Universal qPCR Master Mix is an optimized 2X reaction mix for real-time qPCR detection and quantitation of target DNA sequences using the SYBR/FAM channel of most real-time qPCR instruments. It contains Hot Start Taq DNA Polymerase and has been formulated with a unique passive reference dye that is compatible across a variety of instrument platforms (including those that require a high or low ROX reference signal). It also features dUTP for carryover prevention and a non-fluorescent, visible dye to monitor reaction setup. This dye does not spectrally overlap with fluorescent dyes used for qPCR and will not interfere with real-time detection.
This product is provided in 1 ml volumes sufficient for preparation of 100 qPCR reactions, and is compatible with both enzymatic and sonication-fragmented DNA samples from SimpleChIP® enzymatic and sonication ChIP kits. This master mix formulation is supplied at 2X concentration and contains all PCR components required for amplification and quantitation of DNA, except primers and a DNA template.
This product is stable for 12 months when stored at -20°C and protected from light. It is stable for up to 30 freeze/thaw cycles and can be stored at 4°C, protected from light, for up to 1 month.
Dye-based quantitative PCR (qPCR) uses real-time fluorescence of a double-stranded DNA (dsDNA) binding dye, most commonly SYBR® Green I, to measure DNA amplification as it occurs during each cycle of PCR. At a point where the fluorescence signal is confidently detected over the background fluorescence, a quantification cycle, or CT value, can be determined. CT values can be used to evaluate relative target abundance between two or more samples, or to calculate absolute target quantities in reference to an appropriate standard curve, derived from a series of known dilutions. qPCR is commonly used to detect and quantify target genes in genomic DNA that is enriched by chromatin immunoprecipitation (ChIP).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. SimpleChIP is a registered trademark of Cell Signaling Technology, Inc. SYBR is a registered trademark of Molecular Probes, Inc.
Discover what’s going on at CST, receive our latest application notes and tips, read our science features, and learn about our products.
|88989P||1 ml||$ 65.0|
|88989S||5 ml (5 x 1ml)||$ 290.0|