Sam:LabNotes/Microbione/2009-2-5: Difference between revisions

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>Sam Chiang
>Sam Chiang
Line 29: Line 29:
#Perform the Gel electrophoresis for the amplicons -> check the primer specificity
#Perform the Gel electrophoresis for the amplicons -> check the primer specificity
#Repeat the same reactions using regular PCR and bigger cycles(without SYBR)
#Repeat the same reactions using regular PCR and bigger cycles(without SYBR)
----
=Testing human 18S using regular PCR=
==Objective==
#Check if the 18S primers are specific by agarose gel.
#Using different conc. of human gDNA to optimize the condition.
#Using Taq 2X master mix (New England Biolabs) enzyme which is newly opened.
==Reagents==
#H2O - (Ambion)
#Enzyme - Taq 2X master mix (New England Biolabs)
#Template - diluted human genome DNA (Jurkat cell, New England Biolabs
#18S primer mix (10 uM forward + 10 uM Reverse) -> hS18_212, hS18_306
==Exp. Design==
Template              gDNA (100 ng/uL)    H2O
                ------------------------------
                1/10  1/100  1/1000              Dilution factor
                ------------------------------
Primer: 18S-211  #1    #2      #3        #4    (#1, #2 - replicates)
        18S-306  #5    #6      #7        #8    (#4, #5 - replicates)
==Preparation==
'''gDNA Template dilution''':
Stock solution 100 ug/mL = 100 ng/uL Oligo tube
Dilution from 100 ng/uL ->10 ng/uL (1/10), 1 ng/uL (1/100), 100 pg/uL (1/1000)
==Procedures==
Step1: Preparing master mix
                              1          8+2    rxn
        --------------------------------------------
        H2O                  8.0      80.0      uL
        Taq 2X Master Mix  10.0      100.0      uL
        --------------------------------------------
                            18.0      180.0      uL(180/10=18)
Step2: Transfer 18 uL of Master mix into each of 8 wells in PCR tube-strip
Step3: Add 1 uL primer (h18S-211 or h18S-306) and 1 uL template (gDNA or H2O) as indicated in Exp. design
Step4: Perform regular PCR
        Machine: Bio-Rad 2 blocks- DNA engine
        Program name: Gene59
        94C 2min -> (94C 40 sec -> 59C  40 sec -> 72C  1 min ) x 29 cycle -> 72C 10 min -> 4C Forever

Revision as of 02:08, 6 February 2009

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

  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)




Testing human 18S using regular PCR

Objective

  1. Check if the 18S primers are specific by agarose gel.
  2. Using different conc. of human gDNA to optimize the condition.
  3. Using Taq 2X master mix (New England Biolabs) enzyme which is newly opened.

Reagents

  1. H2O - (Ambion)
  2. Enzyme - Taq 2X master mix (New England Biolabs)
  3. Template - diluted human genome DNA (Jurkat cell, New England Biolabs
  4. 18S primer mix (10 uM forward + 10 uM Reverse) -> hS18_212, hS18_306


Exp. Design

Template              gDNA (100 ng/uL)     H2O
                ------------------------------ 
                1/10   1/100  1/1000              Dilution factor
                ------------------------------
Primer: 18S-211  #1     #2      #3         #4    (#1, #2 - replicates)
        18S-306  #5     #6      #7         #8    (#4, #5 - replicates)


Preparation

gDNA Template dilution: Stock solution 100 ug/mL = 100 ng/uL Oligo tube Dilution from 100 ng/uL ->10 ng/uL (1/10), 1 ng/uL (1/100), 100 pg/uL (1/1000)


Procedures

Step1: Preparing master mix

                             1          8+2     rxn
        --------------------------------------------
        H2O                  8.0       80.0      uL
        Taq 2X Master Mix   10.0      100.0      uL
        --------------------------------------------
                            18.0      180.0      uL(180/10=18)

Step2: Transfer 18 uL of Master mix into each of 8 wells in PCR tube-strip

Step3: Add 1 uL primer (h18S-211 or h18S-306) and 1 uL template (gDNA or H2O) as indicated in Exp. design

Step4: Perform regular PCR

        Machine: Bio-Rad 2 blocks- DNA engine
        Program name: Gene59
        94C 2min -> (94C 40 sec -> 59C  40 sec -> 72C  1 min ) x 29 cycle -> 72C 10 min -> 4C Forever