2nd MDA amplification

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Revision as of 20:17, 2 March 2009 by >Sam Chiang (New page: =2nd MDA amplification for Test2 and Test3 results= ==Objective== *To increase the MDA product for downsteam analysis ==Samples & Materials== *Amplicons from Test2 (Cell line ): B7, C8...)
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2nd MDA amplification for Test2 and Test3 results

Objective

  • To increase the MDA product for downsteam analysis

Samples & Materials

  • Amplicons from Test2 (Cell line ): B7, C8
  • Amplicons from Test3 (Cell lone ): B1, B4

Procedure

  • Set up PCR program (on traditional thermocycler)
  • Thaw the N6 primer, reagents from phi29 kit except enzyme
  • Prepare master mix
                      1 rxn     4.5 rxn
    -----------------------------------
              H2O     147.0    661.5     
       10X buffer      20.0     90.0
        N6 Primer      10.0     45.0
             dNTP       8.0     36.0
     Phi29 Enzyme      10.0     45.0
    ------------------------------------
                      195.0    877.5(uL)  877.5/4.5=195
  • Set up 4 PCR tube on the cool-block, labeled
  • Transfer 5 uL MDA amplicon (test2-B7, test2-C8, test3-B1, test3-b4) to each reaction tubes.
  • Transfer 195 uL Master mix in tubes. Mix well by repeat pipetting.
  • Perform amplification on thermocycler 30C 2hr -> 85C 3 min -> 4C forever