Sam:LabNotes/Microbiome/2009-3-9 exp2

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Multiple PCR against human whole genomoe[edit]

Objective[edit]

  • Based on the previous multiple PCR result, increase the primer concentration for those weak bands
  • Test the effect of (DMSO-like) Q-solution come with Qiagen multiple PCR kit


Material and Samples[edit]

Template: human gDNA (1 ng/uL)

  • Prepare the new primer gourp mix for primer set-1(1X = 10 uL, 2X = 20 uL)
    • Set-1 ("B") Blue: including Y-1(2X), 21-1(2X), the rest primers (1X)
    • Set-1 ("Y") Yellow: 11-1(2X), 10-1(2X), 16-1(2X)
    • Set-1 ("R") Red: Use the old one
    • Set-1 ("G") Green: 18-1(2X), 3-1(2X)
    • Set-1 ("O") Orange: 20-1(2X), 7-1(2X)


Procedures[edit]

    "M": Master mix (no Q-solution)
               1 rxn     6 rxn
    --------------------------
     H2O        15.0     90.0
     Template    5.0     30.0
     Taq2X      25.0    150.0
    --------------------------
                45.0    270.0 (uL) 270/6=45
    "MQ": Master mix (with Q-solution)
               1 rxn     6 rxn
    --------------------------
     Q-sol       5.0     30.0 
     H2O        10.0     60.0
     Template    5.0     30.0
     Taq2X      25.0    150.0
    --------------------------
                45.0    270.0 (uL) 270/6=45
  • Transfer Master mix("M") to 5 pcr tubes in "M" strip
  • Transfer Master mix("MQ") to 5 pcr tubes in "MQ" strip
  • Add different primer in each reaction. Adding order: B -> Y -> R-> G-> O from left to right of each strip
  • Perform reaction in thermocycler with program "MPCR-52" Tm=52, Amplifying 35 Cycles.