Sam:LabNotes/Microbione/2009-2-4
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Testing human 18S using realtime PCR
Objective
- Prepare the primers (human 18S and Bacteria 16S) for in the future MDA experiments as positive control confirmation (The Bac 16S primer is based on given sequecne from Dr. Pillai and Dr. Dowd's lab. The blat results showed these sequence match most of E. coli genome).
- Practice realtime PCR machine operation
- Optimaize primer and template concentration and Tm using EconoTaq 2X master enzyme
Reagents
- H2O - (Ambion, bottled Nuclease-free water) leave in PCR hood for UV decontamination
- Dye - 2X SYBR (working sol.)- diluted from 10,000X (Stock sol.)-> 50X (Stock sol.), only 50X and 2X (diluted) SYBR can be kept in 4 degree
- Enzyme - EconoTaq 2X master - kit
- Template - (10 ng/uL)human genome DNA from Jurkat (Human immortalized T-cell line)
- Primer mix (10 uM forward + 10 uM Reverse)
Exp. Design
Template gDNA H2O -------- --- Primer: 18S-211 #1 #2 #3 (#1, #2 - replicates) 18S-306 #4 #5 #6 (#4, #5 - replicates)
Preparation
Primer diluaiton:
Oligo tube -> add H2O (10 times nmole) uL -> 100 uM 10 uL of 100 uM (forward) + 10 uL of 100 uM (reverse) + 80 uL H2O => Primer mix
gDNA Template dilution:
Stock solution 100 ug/mL = 100 ng/uL Oligo tube 5 uL of 100 ng/uL + 45 uL H2O -> 50 uL of 10 ng/uL
Procedures
Step1: Preparing master mix
1 (6+1) rxn -------------------------------------------- H2O 7.0 49.0 uL SYBR(2X) 1.0 7.0 uL EconoTaq 2X Master 10.0 70.0 uL -------------------------------------------- 18.0 126.0 uL(126/7=18)
Step2: Transfer 18 uL of Master mix into each of 6 individual PCR tubes
Step3: Add 1 uL primer (h18S-211 or h18S-306) and 1 uL template (gDNA or H2O) as indicated in Exp. design
Step4: Perform real-time PCR
Program name: sam020409 94C 2min -> (94C 45 sec -> 58C 2 min -> 72C 1 min -> Plate read) x 20 clcle -> 72C 5 min -> 15C hold
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)