Hello guys ,
I am doing EMSA , and I need to design probes , I have the oligo sequence that is expected to be the binding site for the transcription factor.
However , I don't know what is the length I should order , the binding site is only 8 nucleotides, so should I order only that 8 nucleotides or should I include more ?
Second , should I order sense and antisense for the same nucleotides of interest or only sense strand ?
Third, I am going to label the strand with Biotin using a kit. I saw some people mention using restriction enzymes ? I don't understand why we should use restriction enzyme and how ?
Thank you in advance.
It is important to give careful consideration to the locations and characteristics of primers, probes, and amplicons before starting any real-time PCR experiment. Particularly crucial for primers and probes is ensuring you have an appropriate melting temperature (Tm), which determines the conditions under which these will bind to your target sequence. This article provides general guidelines for designing primers and probes and choosing target locations for amplification.
When calculating melting temperatures using IDT SciTools® applications (e.g., OligoAnalyzer® Tool), be sure to enter the appropriate real-time PCR conditions from your experiment. Commonly used parameters are: 50 mM K+, 3 mM Mg2+, and 0.8 mM dNTPs; however, reaction conditions can vary widely from this, particularly with respect to Mg2+concentration. Therefore, to obtain the Tm values for your specific experimental conditions, it is important that you use your own reaction parameters.
You have a choice of using single-quenched or double-quenched probes. IDT recommends use of double-quenched probes because they provide consistently lower background, resulting in higher signal compared to single-quenched probes. Double-quenched probes that include the IDT ZEN™ molecule as a secondary, internal quencher allow for longer probe lengths to be used in addition to providing strong quenching and increased signal. (Read more about the ZEN quencher in the article, Two Quenchers are Better Than One!,) If designing single-quenched probes, ensure that they are 20−30 bases in length. This will help you to achieve an ideal Tm without increasing the distance between the dye and quencher such that the quencher will no longer optimally absorb the fluorescence of the dye. Design your PCR probes to conform to the following guidelines:
- Location: Ideally, the probe should be in close proximity to the forward or reverse primer, but should not overlap with a primer-binding site on the same strand. Probes can be designed to bind to either strand of the target.
- Melting temperature (Tm): Preferably, probes should have a Tm 6–8°C higher than the primers. If the melting temperature is too low, the percentage of probe bound to target will be low. In this case, the primers may amplify a product, but sensitivity may be compromised as all target sites are not saturated with probe resulting in reduced fluorescence signal that does not truly represent the true amount of target present in the sample.
- Annealing temperature (Ta): The annealing temperature should be set no more than 5°C below the lower primer Tm. Use this as a general guideline, but note that optimization may still be necessary.
- GC content: As with primer sequences, aim for a GC content of 35−65% and avoid a G at the 5’ end to prevent quenching of the 5’ fluorophore.
this link is useful
https://www.researchgate.net/.../Where_do_I_get_oligonucleotide_pr...
regards
Dear Mohammed ,
Thank you for the comprehensive answer on designing the probe.
About the location , how close should be the probe to the forward primer ?
And what do you mean that the probe should not overlap with a primer-binding site on the strand ?
Can you help me for other points I mentioned in my question?
Thank you
Hi Doaa,
for EMSA, I would order probe approximately 40 bases long, having recognition seqence of interest in the middle. Order both sense and antisense oligo and anneal them.
Order your oligos (or one oligo) with Bi label. It is faster, more reliable and probably cheaper, than labeling it yourself using a kit.
Yes, hairpin may cause some troubles during annealing, but generally you design your sequence as it is in real DNA to simulate real conditions.
If you suspect, there is a strong hairpin, do not hurry up during annealing. Het your oligos to 95°C and let it cool to room temperature not faster than 1°C/min (or even slower).
Hi all:
I appreciate the discussion. I need to do radioactive EMSA...I need some pointers as to how to get started. My experimental goal is to see if a transcription factor binds to the promoter region of this gene of interest. I do not have the promoter regions cloned yet but there is another promoter that is available in the lab that I could use to just get this procedure established. How do I label the probe? Thanks
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