Sam:LabNotes/Microbiome/2009-3-13: Difference between revisions

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>Sam Chiang
Line 33: Line 33:
                                       19.0      304.0 (uL)
                                       19.0      304.0 (uL)
      
      
     Add 1 uL template  
     Add 1 uL template (non-dultued MDA amplicon)


*QMPCR programs: QM-PCR52
*QMPCR programs: QM-PCR52

Revision as of 17:26, 16 March 2009

Qiagen MPCR test with Q-solution on MDA L4N6/Tre amplicons

Objectives

  • Test of 20 uL volumn of QMPCR with Q-sol
  • Confirm the amplicons from MDA L4N6/Tre


Samples & Materials

  • Primers: Note: use the un-adjusted primer mixture
    • Blue primers mix: Y-1(122), 21-1(200), 1-1(270), 5-1(413), 22-1(757)


  • Templates: (Amplicon label form MDA reaction)
   Row A: B1 B2 B3 B4 B5 B7 B8
       B: C1 C2 C3 C8 A4 gDNA H2O 
       Note: A4 = p-pos    

       Total= 14 rxns
  • Master solution of 20 uL QMPCR with Q-sol.
                                    1 rxn      16 rxn 
    -------------------------------------------------
    RNAse-free H2O                    5.0        80.0  
    Q-solution                        2.0        32.0
    Primer mix (Blue)                 2.0        32.0
    Qiagen kit enzyme (2X)           10.0       160.0
    -------------------------------------------------
                                     19.0       304.0 (uL)
   
    Add 1 uL template (non-dultued MDA amplicon)
  • QMPCR programs: QM-PCR52
     Step1   95C, 15m
     Step2   94C, 30s
     Step3   52C, 1m30s
     Step4   72C, 1m30s
     Step5   Goto Step2 X 30 
     Step6   72C, 10m
     Step7   4C, forever

Results

File:ZhangLab 2 2009-03-13 10hr 09min-with annotate.bmp

  • Gel Electrophoresis
    • 2 % agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe) -> two medium tray with 13-well comb
    • Loading: Sample 10 uL + 2 uL loading buffer; Diluted ladder 10 uL
    • Run at 135 V for 30 min.


Discussion

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