Sam:LabNotes/Microbione/2009-2-5
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Follow up: Testing human 18S 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
- The amplification curves are not distinguishable (Sample vs. Blank) within 20 clycles.
- The number of amplifications cycles we used is not enough?
- The amplifications are not working? (Primers' specificity? Concentration of templates?)
Suggestion
- Perform the Gel electrophoresis for the amplicons -> check the primer specificity
- Repeat the same reactions using regular PCR and bigger cycles(without SYBR)