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

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>Sam Chiang
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>Sam Chiang
No edit summary
Line 13: Line 13:
#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
     Product size: ~200 bp; ~300 bp
     Product size: ~200 bp; ~300 bp
     Annealing Temp: Min:57, Opt:58, Max:59
     Annealing Temp: Min:57, Opt:58, Max:59
 
#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
Line 24: Line 24:
     >h18S_211_r
     >h18S_211_r
     CATTATTCCTAGCTGCGGTA
     CATTATTCCTAGCTGCGGTA
 
     -----------------------------------------------
    OLIGO            start  len     tm    gc%  any    3' seq
     >h18S_306_f
     LEFT PRIMER        758  20  57.86  40.00  6.00  2.00 TTGCTGCAGTTAAAAAGCTC
     GTACAGTGAAACTGCGAATG
     RIGHT PRIMER      968  20  57.56  45.00  4.00  2.00 CATTATTCCTAGCTGCGGTA
     >h18S_306_r
     SEQUENCE SIZE: 1969
     CGACTACCATCGAAAGTTGA
     INCLUDED REGION SIZE: 1969
     -----------------------------------------------
     PRODUCT SIZE: 211, PAIR ANY COMPL: 5.00, PAIR 3' COMPL: 1.00

Revision as of 06:38, 9 February 2009

Primer design for human 18S and Bacteria 16S

Objective

Besides 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

  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
  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
    -----------------------------------------------