Do you know any method to distinguish small target band of primer dimer?
( They have similar size.)
I tried higher concentration of agarose gel and longer electrophoresis run time. but i didn`t see distinct bands. my sequence doesn`t have any restriction enzyme site for enzymes I can access.
Primer dimers may be visible after gel electrophoresis of the PCR product. PDs in ethidium bromide-stained gels are typically seen as a 30-50 base-pair (bp) band or smear of moderate to high intensity and distinguishable from the band of the target sequence, which is typically longer than 50 bp.
In quantitative PCR, PDs may be detected by melting curve analysis with intercalating dyes, such as SYBR Green I, a nonspecific dye for detection of double-stranded DNA. Because they usually consist of short sequences, the PDs denature at lower temperature than the target sequence and hence can be distinguished by their melting-curve characteristics.
either use PAGE gels or get rid of the primer dimer by using either a hot start taq polymerase or different primers that do not produce a dimer
Primer dimers may be visible after gel electrophoresis of the PCR product. PDs in ethidium bromide-stained gels are typically seen as a 30-50 base-pair (bp) band or smear of moderate to high intensity and distinguishable from the band of the target sequence, which is typically longer than 50 bp.
In quantitative PCR, PDs may be detected by melting curve analysis with intercalating dyes, such as SYBR Green I, a nonspecific dye for detection of double-stranded DNA. Because they usually consist of short sequences, the PDs denature at lower temperature than the target sequence and hence can be distinguished by their melting-curve characteristics.
what size is your amplimer and how long are the primers? Perhaps the answer is to enlarge the amplimer if you just need it for sequencing or cloning in the next step of your work. More details would get you better answers
Hello!
You can detect pcr product in this case by real time pcr using Tagman. This is the easiest way.
Well, my idea is quite costly, but there you have it:
It is based on the condition of CCGG sequence being present betwen the primer sequences in the desired PCR product.
1. Run the PCR reaction with 5-Methylcytosine dNTP Mix.
2. Incubate the resulting DNA with HpaII restrictase. This will cleave the desired PCR product into smaller dsDNA strands.
The primer dimer products will stay in one piece even if they contain the CCGG sequence.
Because there is no 5-Methylcytosine in the primers.
Hi..You can use probe to differentiate amplified product from primer dimer.
Have you tried Melt Curve Analysis?
Hello there!
I, personally, use DMSO (DiMethylSulfOxide) to purify my electrophoresis results. However, if you have low quality or weak bods, you may not use DMSO as it will make those expected bods disappear as well.
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