2nd MDA amplification

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2nd MDA amplification for Test2 and Test3 results[edit]

Objective[edit]

  • To increase the MDA product for downsteam analysis


Samples & Materials[edit]

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


Procedure[edit]

  • 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


PCR confirmation[edit]

  • Primer: 18S, Primer 2-1
  • Template: 1/10 dilution of 2nd MDA amplicons (B7, C8, B1, B4), Pos (1 ng/uL gDNA), H2O
  • PCR Master mix x 2 (Primer various)
       1 rxn    7 rxns
  H2O    3.5      24.5
  Primer 0.5       3.5 
  Taq2X  5.0      35.0
  --------------------
         9.0      63.0 uL 63/7=9 -> Template 1 uL
  • Set up PCR reaction on the cool-block
    • Since only one Thermocycler block is available (others are occupied, we use the 56C Tm for both two reaction which acturally need 52C and 59C for Tm)


Gel electrophoresis[edit]

  • 1.5% agarosel (100 mL + 8 uL SYBR safe), 135V, run 20 min.
  • Loading order: T2B7, T2C8, T3B1, T3B4, Pos, H2O (Left to Right)


Results[edit]

File:ZhangLab 2 2009-03-01 10hr 33min-p18-p2-1.bmp

  • Left: Test by 18S primer (306 bp). Right: Test by 2-1 primer (540 bp)
  • Some bands are very weak probably due to the inappropriate Tm we used.
  • We will give T2B7 and T3B1 samples to Illumina for analysis.