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='''Primer design for human 18S and Bacteria 16S'''= ==Objective== Beside the realtime monitoring, #Design 18S primers for positive control / validation purpose of a successful MDA reaction using human genome template. #Design 16S primers for positive control / validation purpose of a successful MDA reaction using bacteria genome template. #These primers could also be used to detect the contamination of exogenus gDNA. ==Human S18 primer== #Collect the human S18 cDNA sequence #Clean the sequcne and mask the inconsistat nucleotide #Paste the sequence on KZ's Primer3 calculator[http://arep.med.harvard.edu/kzhang/cgi-bin/primer3_www.cgi] #Criteria: Primer size: Min: 18bp, opt:20 bp, Max: 22bp Product size: ~200 bp; ~300 bp Annealing Temp: Min:57, Opt:58, Max:59 '''Results''' #Using Netprimer[http://www.premierbiosoft.com/netprimer/index.html]to evaluate primer structure. #Pick up two primers with differnt size of amplicon. >h18S_211_f TTGCTGCAGTTAAAAAGCTC >h18S_211_r CATTATTCCTAGCTGCGGTA ----------------------------------------------- >h18S_306_f GTACAGTGAAACTGCGAATG >h18S_306_r CGACTACCATCGAAAGTTGA ----------------------------------------------- *[[Media:Human 18S rRNA gene primer design.doc|Human 18S rRNA gene primer design]] ==Bacteria S16 primer== #Use the reported 16S primers which has been tested on conserved region of 16S gene[[Media:Soni_E coli 0157 and AI-2_2008.PDF| (citaiton of 16S primer)]]. #Primer sequences have been test on '''UCSD prokaryote genome browser'''[http://archaea.ucsc.edu/cgi-bin/hgGateway?org=Salmonella+typhimurium+LT2&db=salmTyph_LT2&hgsid=182679]. All of primers were able to match most E. coli stains' 16S gene region. '''Primer sequences''' >Pilli_16S_f CCAGCAGCCGCGGTAAT >Pilli_16S_r TGCGCTTTACGCCCAGTAAT ---------------------------------------------- >Dowd_16S-1-f TCCTACGGGAGGCAGCAGT >Dowd_16S-1-r GGACTACCAGGGTATCTAATCCTGTT ---------------------------------------------- >Dowd_16S-2-f CGCTAGTAATCGTGGATCAGAATG >Dowd_16S-2-r TGTGACGGGCGGTGTGTA *[[Media:16S primers.doc|16S primers condition]]
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