Sam:LabNotes/Human/2009-4-12

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Revision as of 18:35, 13 April 2009 by >Sam Chiang (→‎Procedure)
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Realtime PCR validation for L4N9 (3/31/'09) and N6 (04/01/'09) MDA amplicons

Procedure

  • Experiment design
    • Primers: 1-1, 2-1, 3-1, 4-1, 5-1, 6-1
    • Templates: Candidates MDA amplicon. 7 samples from 3/31, 7 samples from 4/01, pos (gDNA 100pg/uL), NTC(H2O)
  • Thaw gDNA(100pg/uL), Taq 2X enzyme, primers mix and diluted templates (MDA amplicon in 1/50 dilution)
  • Set up realtime PCR program (decribed as following)
  • Set up 12 PCR strip tubes on cool block, labed as column 1 to column 12.
  • Expemriment arrangements
            Column 1  ->  6        Column 7  -> 12
            Primer 1-1->6-1        Primer 1-1->6-1
                B6(4-01)              B7(4-01)      
                D2(4-01)              D4(4-01)  
                D5(4-01)              D7(4-01)  
                D8(4-01)              B3(3-31)  
                B4(3-31)              B6(3-31)  
                C2(3-31)              C5(3-31)  
                C7(3-31)              C8(3-31)  
                POS(100pg)            NTC  
  • Prepare diluted primers in PCR tubes
                           1rxn  (8+1)x2 ->20rxns
  Primer mix (f+r 10 uM)    1.0     20.0 uL
  H2O                       7.0    140.0 uL
  ----------------------------------------
                            8.0    160.0
  • Transfer 8 uL diluted primer into each reaction based on the arrangements
  • Transfer 2 uL diluted templates into each reaction based on arrangements
  • Prepare master mix in 1.5-mL microcentrifuge tube
               1rxn  (12+2)x8=112 rxns
  Taq (2X)      9.9         1108.8
  SYBR (50X)    0.1           11.2 
  --------------------------------
               10.0         1120.0
  • Mix well the master mix by vortexing. Seperate master into 8 PCR tubes then transer 10 uL Master mix into each reaction using multichannel pipettor.
  • Gently vortex and spin down the PCR reaction tubes. Place in Chromo4 machine immediately to perform realtime PCR reaction.
  • Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C sec -> plate read) x 45 -> Melting curve (52C->94C, read every 2C, hold 1min) -> 15C forever

Results

Primer 1-1 ' ' ' '
Content Description C(t) dCT(NTC-Sample) Melting curve matched to Pos control
Sample B6 (4/1) N6-No-Tre 37.91 -1.06 n
Sample B7 (4/1) N6-No-Tre 7.72 29.13 n
Sample D2 (4/1) N6-No-Tre 22.07 14.78 n
Sample D4 (4/1) N6-No-Tre 6.37 30.48 n
Sample D5 (4/1) N6-No-Tre 6.77 30.08 n
Sample D7 (4/1) N6-No-Tre 7.37 29.48 n
Sample D8 (4/1) N6-No-Tre 5.48 31.37 n
Sample B3 (3/31) L4N9-Tre 36.3 0.55 y
Sample B4 (3/31) L4N9-Tre 33.25 3.6 y
Sample B6 (3/31) L4N9-Tre 34.64 2.21 n
Sample C2 (3/31) L4N9-Tre 30.49 6.36 y
Sample C5 (3/31) L4N9-Tre 4.76 32.09 n
Sample C7 (3/31) L4N9-Tre 31.67 5.18 y
Sample C8 (3/31) L4N9-Tre 32.3 4.55 n
Sample pos 24.3
Blank ntc 36.85