Sam:LabNotes/Microbiome/2009-3-11: Difference between revisions

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==Results==
==Results==
[[Image:ZhangLab_2 2009-03-12 10hr 09min-with annotate.bmp|ZhangLab_2 2009-03-12 10hr 09min-with annotate|px600]]
[[Image:ZhangLab_2 2009-03-12 10hr 09min-with annotate.bmp|px600]]

Revision as of 19:21, 12 March 2009

PCR confirmation for human MDA amplicons & Q-solution test on Qiagen MPCR kit

Objectives

  • Compare if the Q-solution can improve the results using Qiagen MPCR kit
  • Pick up more candidates of amplicons to be tested using the Taq2X MPCR with set-up condition


Samples & Materials

  • Primers:
    • Blue primers mix: Y-1(122), 21-1(200), 1-1(270), 5-1(413), 22-1(757)
    • Yello primers mix: 11-1(131), 10-1(214), 15-1(297), 17-1(419), 16-1(806)
  • Templates
    • For all Qiagen MPCR (QMPCR) tests: 5 uL of 1 ng/uL human gDNA
    • For TaqPol2X MPCR (TMPCR) tests: 1 uL of Amplicon candidates picked up from human MDA (03/10/09): B10, E9, E10, F9, F10, H8, H9, H10


Procedures

  • QMPCR - no Q-solution
                            1 rxn      
    -----------------------------
    RNAse-free H2O           15.0 
    Template (1 ng/uL)        5.0   
    Qiagen kit enzyme (2X)   25.0 
    -----------------------------
                             45.0 (uL) 
    Add 5 uL primer mix Bule / Yellow (Use the unadjusted set -> equal ammount of each pair)
  • QMPCR - with Q-solution
                            1 rxn 
    -----------------------------
    RNAse-free H2O           10.0 
    Q-solution                5.0
    Template (1 ng/uL)        5.0   
    Qiagen kit enzyme (2X)   25.0 
    -----------------------------
                             45.0 (uL) 
    Add 5 uL primer mix Bule / Yellow (Use the unadjusted set -> equal ammount of each pair)
  • TMPCR
                            1 rxn       
    -----------------------------
    RNAse-free H2O           19.0 
    Primer Bule               5.0 (Use the djusted set)
    NEB Taq pol.(2X)         25.0 
    -----------------------------
                             49.0 (uL)
    Add 1 uL MDA amplicon  


  • QMPCR programs: QM-PCR52 Note: cycles were decreased from 35 to 30
     Step1   95C, 15m
     Step2   94C, 30s
     Step3   52C, 1m30s
     Step4   72C, 1m30s
     Step5   Goto Step2 X 30 
     Step6   72C, 10m
     Step7   4C, forever
  • QMPCR programs: TM-PCR52 Note: cycles were increaded from 35 to 40
     Step1   94C, 2m
     Step2   94C, 40s
     Step3   52C, 40s
     Step4   72C, 1ms
     Step5   Goto Step2 X 40 
     Step6   72C, 10m
     Step7   4C, forever

Results

px600