Sam:LabNotes/Microbione/2009-2-3: Difference between revisions
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==Bacteria S16 primer== | ==Bacteria S16 primer== | ||
#Use the reported 16S primers which has been tested on conserved region of 16S gene. | #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[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' S16 gene region. | #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' 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 | |||
-------------------- | |||
* |
Revision as of 07:09, 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 --------------------