Sam:LabNotes/Human/2009-5-10 exp1

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Perform 2nd round MDA on the amplicons from 200pg L4N9 [edit]

Objective[edit]

  • Amplify the MDA amplicons from 200pg-L4N9, L4N8, L4N7 for Illumina Infinium array genotyping
  • The request DNA concentration for Infinium assay: 5 ug/uL for 15 uL
  • In order to optimize the condition of DNA purification, only L4N9 was tested with different reaction time (4h vs 8h)


Procedure[edit]

  • Set up PCR program on traditional thermocycler
    • MDA4h 30C 4hr -> 85C 3 min -> 4C forever
    • MDA8h 30C 8hr -> 85C 3 min -> 4C forever
  • Thaw the L4N9 primer, reagents from phi29 kit except enzyme
  • Set up reaction in PCR tubes
                  4hr-rxn/8hr-rxn
    -----------------------
              H2O     147.0   
       10X buffer      20.0    
       L4N9 Primer     10.0     
             dNTP       8.0     
     Phi29 Enzyme      10.0     
    -----------------------
                      195.0   
  • Transfer 5 uL MDA amplicon, amplicons from 200pg-L4N9, into each of two reactions. Mix well by repeat pipetting.
  • Perform amplification on thermocycler based on assigned program (MDA4h/MDA8h)