I found in articles primers specific to different strains eg: Akkermancia muciniphila ,Bacteroides vulgatus... I noticed that many people still check these primers on blast to ensure their qualities and their specificities. Who can give me the steps to follow on to check primers on blast tool? I give you an example (Enterobacteriaceae)
EntF: 5'CATTGACGTTACCCGCAGAAGAA3'
EntR: 5'CGCTTGCACCCTCCGTATTA3'
Thanks
You can BAST in NCBI BLAST page using Nucleotide BLAST. You can BLAST combined or individual primer. In result page check for any other position for primer binding.
Dear Meriem
I guess you wish to find how specific would be your primers.
The first thing you do is to Primer-blast in checking the desired hits: http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Second, to find how specific they are, please use the raw DNA sequences (the entire genomic context), that will allow the tool to differntiate between the off-targets and the template you are using. If you get the hit, then your f and r primers should be in the list.
Third, please don't forget the option "Primer Pair Specificity Checking"
Best
Prash
One of the best way using online insilico PCR tools.
this is one the best sites: http://insilico.ehu.es/
Go to this site and check your primer specificity with any bacterial species.
I have no much experience on primer design, please would you help me to choose the best pair from this list? and why?
I will be great for explanation
Primer pair 1
Sequence (5'->3')Template strandLengthStartStopTmGC%Self complementaritySelf 3' complementarityForward primerCGCGCGCTATAAATGCAACTPlus2065167060.0450.006.002.00Reverse primerACCAGCGGGCTGTTATACTGMinus2078876959.8255.006.002.00Product length138
Primer pair 2
Sequence (5'->3')Template strandLengthStartStopTmGC%Self complementaritySelf 3' complementarityForward primerAAAATTTCATAGCCGCCCGCPlus2048049959.9050.006.003.00Reverse primerATTTTCACGTTGCCGCTCTGMinus2063261359.7650.004.001.00Product length153
Primer pair 3
Sequence (5'->3')Template strandLengthStartStopTmGC%Self complementaritySelf 3' complementarityForward primerTTTCATCATGATCCGCCGGTPlus2044846759.8250.008.003.00Reverse primerGGTGGTAATCAGGCCTTCGTMinus2070268359.7555.006.002.00Product length255
Primer pair 4
Sequence (5'->3')Template strandLengthStartStopTmGC%Self complementaritySelf 3' complementarityForward primerCAGACCGCGCGCTATAAATGPlus2064666559.8455.006.000.00Reverse primerGCCAGCGGAAACGGAATATGMinus2084882959.6955.004.002.00Product length203
Primer pair 5
Sequence (5'->3')Template strandLengthStartStopTmGC%Self complementaritySelf 3' complementarityForward primerGCATTGGCACCGATGATAGCPlus2022724659.7655.006.002.00Reverse primerAGCGCGCCAGAATAAAATGGMinus2037635759.6250.006.002.00Product length150
Primer pair 6
Sequence (5'->3')Template strandLengthStartStopTmGC%Self complementaritySelf 3' complementarityForward primerATAGCTGCACCCATCCGTTTPlus2043145059.7450.006.000.00Reverse primerTCGGTGGTAATCAGGCCTTCMinus2070468559.4655.006.002.00Product length274
Primer pair 7
Sequence (5'->3')Template strandLengthStartStopTmGC%Self complementaritySelf 3' complementarityForward primerTAGCTGCACCCATCCGTTTCPlus2043245160.3955.004.000.00Reverse primerGGCGGCATATGCACTTCAATMinus2058156259.3350.008.003.00Product length150
Primer pair 8
Sequence (5'->3')Template strandLengthStartStopTmGC%Self complementaritySelf 3' complementarityForward primerACGAAGGCCTGATTACCACCPlus2068370259.7555.006.000.00Reverse primerTAGTTCGGTTCTTCGCCCATMinus2098096159.1050.002.002.00Product length298
Primer pair 9
Sequence (5'->3')Template strandLengthStartStopTmGC%Self complementaritySelf 3' complementarityForward primerTGATTGGACCAAACTGCGCTPlus2024926860.5450.007.002.00Reverse primerGATGAAACGGATGGGTGCAGMinus2045443559.2755.004.002.00Product length206
Primer pair 10
Sequence (5'->3')Template strandLengthStartStopTmGC%Self complementaritySelf 3' complementarityForward primerCATAGCTGCACCCATCCGTTPlus2043044960.4655.006.001.00Reverse primerAATTTTCACGTTGCCGCTCTMinus2063361459.0545.004.000.00Product length204
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