Sam:LabNotes/Microbione/2009-2-3: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Sam Chiang
No edit summary
>Sam Chiang
 
(16 intermediate revisions by the same user not shown)
Line 4: Line 4:


==Objective==
==Objective==
Besides the realtime monitoring,
Beside the realtime monitoring,
#Design 18S primers for positive control / validation purpose of a successful MDA reaction using human genome template.
#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.
#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.
#These primers could also be used to detect the contamination of exogenus gDNA.


==Human S18 primer==
==Human S18 primer==
#Collect the human S18 cDNA sequence
#Collect the human S18 cDNA sequence
#Clean the sequcne and mask the inconsistat nucleotide
#Clean the sequcne and mask the inconsistat nucleotide
#Paste the sequence on KZ's Primer3 calculator[http://www.example.com link title]
#Paste the sequence on KZ's Primer3 calculator[http://arep.med.harvard.edu/kzhang/cgi-bin/primer3_www.cgi]
#Criteria:
#Criteria:
     Primer size: Min: 18bp, opt:20 bp, Max: 22bp
     Primer size: Min: 18bp, opt:20 bp, Max: 22bp
Line 19: Line 18:
     Annealing Temp: Min:57, Opt:58, Max:59
     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.
#Pick up two primers with differnt size of amplicon.
     >h18S_211_f
     >h18S_211_f                
     TTGCTGCAGTTAAAAAGCTC
     TTGCTGCAGTTAAAAAGCTC      
     >h18S_211_r
     >h18S_211_r
     CATTATTCCTAGCTGCGGTA
     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.


    OLIGO            start  len      tm     gc%  any    3' seq
'''Primer sequences'''
    LEFT PRIMER        758  20  57.86  40.00  6.00  2.00 TTGCTGCAGTTAAAAAGCTC
    >Pilli_16S_f
    RIGHT PRIMER      968  20  57.56  45.00  4.00  2.00 CATTATTCCTAGCTGCGGTA
    CCAGCAGCCGCGGTAAT
    SEQUENCE SIZE: 1969
    >Pilli_16S_r
    INCLUDED REGION SIZE: 1969
    TGCGCTTTACGCCCAGTAAT
    PRODUCT SIZE: 211, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 1.00
    ----------------------------------------------
    >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]]

Latest revision as of 18:34, 9 February 2009

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 UCSD 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