Sam:LabNotes/Microbione/2009-2-4: Difference between revisions

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=Testing human 18S and Bac (E. coli) 16S primers using realtime PCR=
=Testing human 18S using realtime PCR=






==Objective==
==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).
#Prepare the primers (human 18S and Bacteria 16S) for the future use in 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). Primers info are referred to [[Sam:LabNotes/Microbione/2009-2-3]]
#Practice realtime PCR machine operation
#Practice realtime PCR machine operation
#Optimaize primer and template concentration and Tm using EconoTaq 2X master enzyme
#Optimaize primer and template concentration and Tm using EconoTaq 2X master enzyme


==Reagents==
==Reagents==
Line 35: Line 34:




==Procedures:==
==Procedures==


Step1: Preparing master mix
Step1: Preparing master mix
                               1 rxn    (6+1)   rxn
                               1       (6+1)     rxn
         --------------------------------------------
         --------------------------------------------
         H2O                  7.0      49.0      uL
         H2O                  7.0      49.0      uL
Line 48: Line 47:
Step2: Transfer 18 uL of Master mix into each of 6 individual PCR tubes
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 in Exp. design
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  
Step4: Perform real-time PCR  
         Program name: sam020409
         Program name: sam020409
         94C 2min -> (94C 45 sec -> 58C 2 min -> 72C 1 min -> Plate read) x 20 clcle -> 72C 5 min -> 15C hold
         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)'''
[[Image:sam020409.jpg|600px|none]]
'''FIG.2 Realtime amplification plot (with log in flourescent)'''
[[Image:sam020409-log.jpg|600px|none]]
==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)

Latest revision as of 07:20, 9 February 2009

Testing human 18S using realtime PCR[edit]

Objective[edit]

  1. Prepare the primers (human 18S and Bacteria 16S) for the future use in 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). Primers info are referred to Sam:LabNotes/Microbione/2009-2-3
  2. Practice realtime PCR machine operation
  3. Optimaize primer and template concentration and Tm using EconoTaq 2X master enzyme

Reagents[edit]

  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[edit]

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


Preparation[edit]

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[edit]

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[edit]

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


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


Discussion[edit]

  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[edit]

  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)