Sam:LabNotes/Microbione/2009-2-4

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Revision as of 17:35, 8 February 2009 by >Sam Chiang
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Testing human 18S using realtime PCR

Objective

  1. 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).
  2. Practice realtime PCR machine operation
  3. Optimaize primer and template concentration and Tm using EconoTaq 2X master enzyme


Reagents

  1. H2O - (Ambion, bottled Nuclease-free water) leave in PCR hood for UV decontamination
  2. 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
  3. Enzyme - EconoTaq 2X master - kit
  4. Template - (10 ng/uL)human genome DNA from Jurkat (Human immortalized T-cell line)
  5. 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

  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)