Sam:LabNotes/Microbiome-new/2009-2-3

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Primer design for human 18S and Bacteria 16S[edit]

Objective[edit]

Beside the realtime monitoring,

  1. Design 18S primers for positive control / validation purpose of a successful MDA reaction using human genome template.
  2. Design 16S primers for positive control / validation purpose of a successful MDA reaction using bacteria genome template.
  3. These primers could also be used to detect the contamination of exogenus gDNA.

Human S18 primer[edit]

  1. Collect the human S18 cDNA sequence
  2. Clean the sequcne and mask the inconsistat nucleotide
  3. Paste the sequence on KZ's Primer3 calculator[1]
  4. 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

  1. Using Netprimer[2]to evaluate primer structure.
  2. 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
    -----------------------------------------------


Bacteria S16 primer[edit]

  1. Use the reported 16S primers which has been tested on conserved region of 16S gene (citaiton of 16S primer).
  2. Primer sequences have been test on UCSC prokaryote genome browser[3]. 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