Sam:LabNotes/Microbiome-new/2009-2-3
Jump to navigation
Jump to search
Primer design for human 18S and Bacteria 16S[edit]
Objective[edit]
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[edit]
- Collect the human S18 cDNA sequence
- Clean the sequcne and mask the inconsistat nucleotide
- Paste the sequence on KZ's Primer3 calculator[1]
- 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[2]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 -----------------------------------------------
Bacteria S16 primer[edit]
- Use the reported 16S primers which has been tested on conserved region of 16S gene (citaiton of 16S primer).
- 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