Sam:LabNotes/Human/2009-4-12

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Revision as of 21:02, 12 April 2009 by >Sam Chiang (New page: ='''Realtime PCR validation for L4N9 (3/31/'09) and N6 (04/01/'09) MDA amplicons'''= ==Procedure== *Experiment design Primer 1-1->6-1 Primer 1-1->6-1 Column ...)
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Realtime PCR validation for L4N9 (3/31/'09) and N6 (04/01/'09) MDA amplicons

Procedure

  • Experiment design
            Primer 1-1->6-1        Primer 1-1->6-1
 Column         B6(3-31)              B7(3-31)      
  • Thaw following regents:
    • Taq pol.(2X)
    • Primer Tamplate
  • Primers (4 uL)
 10 uL (10 mM of F + R) + 70 uL H2O mixture
                      1 rxn       20 rxn
    Primer (f+r)        1.0        10 uL
    H2O                 7.0        70 uL
    ------------------------------------
                        8.0        80 uL
  
    Place diluted primer in eight PCR tubes(column 1 to 8 (left->right))
    1-1, 2-1, 3-1, 4-1, 1-1, 2-1, 3-1, 4-1
  • Aliquots Taq2X enzymes into PCR tubes (100 uL) x 8 PCR tubes
  • Preparing PCR strip tubes
  • Using multi-channel pipette to upload all reagents
    • Enzyme (5 uL): Taq Mix 2X
    • Primer (4 uL)
  • Using p2.5 pipettor to add diluted (1/50) Templates: (1 uL)
  RowA (left) 3-31 B3,  RowA(right) 3-31 B4
  RowB (left) 3-31 B6,  RowB(right) 3-31 C2
  RowC (left) 3-31 C5,  RowC(right) 3-31 C7
  RowD (left) 3-31 C8,  RowD(right) 3-31 gDNA 3 ng
  • Program: 52T35C (Tm=52C, for 35 cycles
  • Gel electrophoresis
    • 2% agarose gel, midium tray with 26-well comb
    • Sample (8uL) + Loading buffer (1.5 uL)
    • Run at 135V for 20 min

Results

 File:ZhangLab 2 2009-04-01 14hr 12min-annotate.jpg 
 File:ZhangLab 2 2009-04-01 14hr 52min-annotate.jpg
 
 Primer(amplicon size)
 
 1-1(270 bp), 2-1(540 bp), 3-1(235 bp), 4-1(383 bp)