Sam:LabNotes/Microbione/2009-2-3: Difference between revisions
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>Sam Chiang |
>Sam Chiang No edit summary |
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Line 44: | Line 44: | ||
>Pilli_16S_r | >Pilli_16S_r | ||
TGCGCTTTACGCCCAGTAAT | TGCGCTTTACGCCCAGTAAT | ||
-------------------- | ---------------------------------------------- | ||
>Dowd_16S-1-f | >Dowd_16S-1-f | ||
TCCTACGGGAGGCAGCAGT | TCCTACGGGAGGCAGCAGT | ||
>Dowd_16S-1-r | >Dowd_16S-1-r | ||
GGACTACCAGGGTATCTAATCCTGTT | GGACTACCAGGGTATCTAATCCTGTT | ||
-------------------- | ---------------------------------------------- | ||
>Dowd_16S-2-f | >Dowd_16S-2-f | ||
CGCTAGTAATCGTGGATCAGAATG | CGCTAGTAATCGTGGATCAGAATG | ||
Line 55: | Line 55: | ||
TGTGACGGGCGGTGTGTA | TGTGACGGGCGGTGTGTA | ||
-------------------- | -------------------- | ||
* | *[[Media:16S primers.doc|16S primers condition]] |
Revision as of 07:10, 9 February 2009
Primer design for human 18S and Bacteria 16S
Objective
Besides 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[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
- Use the reported 16S primers which has been tested on conserved region of 16S gene.
- Primer sequences have been test on UCSD prokaryote genome browser[3]. All of primers were able to match most E. coli stains' S16 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 --------------------