Characteristics: product length, GC content, base repeats, melting temperature, exon-exon junction vs including an intron, self and 3' complementarity, the specificity of the primers, primer length, GC clamp, etc....)
Hi Ohanes Haroutoun Ashekyan
First of all let's decide which goal do you consider.
Gene expression?
SNP detection?
Methyl Specific PCR?
Sanger sequencing?
Work with microRNA
etc... even custom NGS panel design )
And which chemistry do you plan ti use:
TaqMan (I prefer)
SYBR Green
other?
In any case for me - the best way is https://bioinfo.ut.ee/primer3-0.4.0/ and after that IDT olygoanalyser (free but with registration).
Regarding primers...I need more start information to help, please do not hecitate to write.
hi,
-Primer designing tools: PrimerBLAST, IDT Primerquest
-Primer length: ~20
-Product length: 100-150bp
-GC content: >50%
-Tm= 50-60 deg. C
- Exon junction span: exon-exon junction
-minimum self and 3' complementarity
cheers!!!
Hi Ohanes Haroutoun Ashekyan
First of all let's decide which goal do you consider.
Gene expression?
SNP detection?
Methyl Specific PCR?
Sanger sequencing?
Work with microRNA
etc... even custom NGS panel design )
And which chemistry do you plan ti use:
TaqMan (I prefer)
SYBR Green
other?
In any case for me - the best way is https://bioinfo.ut.ee/primer3-0.4.0/ and after that IDT olygoanalyser (free but with registration).
Regarding primers...I need more start information to help, please do not hecitate to write.
It really depends on what you're doing, but generally I use primer3 and check then check for secondary structure with the IDT oligoanalyzer tool.
Use the Oligoanalyzer to make sure the Tm of any secondary structure and self-dimer is much lower than your annealing temp (I don't believe Primer 3 checks for secondary structure, but could be wrong.)
The Tm should be around 60C- but it's more important that the Tms of both of your primers are within around 5C.
When considering product length, just make sure that you set your extension time (72C in the cycling parameters) long enough that it can make the entire product.
Andrei S. Babenka it is for gene expression. I also want your advice on chemistry use. Thank you for the information.
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