Sam:LabNotes/Microbione/2009-2-5

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Revision as of 18:37, 8 February 2009 by >Sam Chiang (→‎Exp. Design)
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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 bp  #1     #2      #3         #4    (#1, #2 - replicates)
        18S-306 bp  #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 

Step5: Agarose gel electrophoresis condition

        1.5% agarose gel: 0.75 g + 50 mL (0.5 TBE). Heat up with 30 sec + 20 sec 
        Gel stain: 4uL SYBR safe (Invitrogen)/ 50 mL Agarose gel mix
        Small gel tray with 8-well comb 
        Ladder: Low mass ladder (Invitrogen)
        Sample loading: (6 uL sample + 1 uL dye) -> load 6 uL mix
        Loading order: L->R,  #1~#8 (02-05-09' PCR products) -> #1~#6(02-04-09' PCR products) -> ladder       
        Running at 135 V for 30 min.

Results

FIG. xxxxxxxxxxxxxxxxx

Discussion


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