Sam:LabNotes/Microbione/2009-2-5

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Revision as of 21:40, 5 February 2009 by >Sam Chiang (→‎Suggestion)
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Follow up: Testing human 18S and Bac (E. coli) 16S primers using realtime PCR

Exp. Design

Template         gDNA      H2O
                --------  ---
Primer: 18S-211  #1   #2   #3    (#1, #2 - replicates)
        18S-306  #4   #5   #6    (#4, #5 - replicates)


Results

FIG.1 Realtime amplification plot (no log in flourescent)


FIG.2 Realtime amplification plot (with log in flourescent)


Discussion

  1. The amplification curves are not distinguishable (Sample vs. Blank) within 20 clycles.
    1. The number of amplifications cycles we used is not enough?
    2. The amplifications are not working? (Primers' specificity? Concentration of templates?)


Suggestion

  1. Perform the Gel electrophoresis for the amplicons -> check the primer specificity
  2. Repeat the same reactions using regular PCR and bigger cycles(without SYBR)